Rapafucin derivative compounds and methods of use thereof

ABSTRACT

The present disclosure provides macrocyclic compounds inspired by the immunophilin ligand family of natural products FK506 and rapamycin. The generation of a Rapafucin library of macrocyles that contain FK506 and rapamycin binding domains should have great potential as new leads for developing drugs to be used for treating diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 16/590,087 filed Oct. 1, 2019, now pending; which is acontinuation-in-part application of U.S. application Ser. No. 16/074,017filed Jul. 30, 2018, now issued as U.S. Pat. No. 10,662,220; which is a35 USC § 371 National Stage Application of International Application No.PCT/US2017/016481 filed Feb. 3, 2017, now expired; which claims thebenefit under 35 USC § 119(e) to U.S. Application Ser. No. 62/291,437filed Feb. 4, 2016, now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. CA174428awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND INFORMATION

The macrocyclic natural products FK506 and rapamycin are approvedimmunosuppressive drugs with important biological activities. Both havebeen shown to inhibit T cell activation, albeit with distinctmechanisms. In addition, rapamycin has been shown to have stronganti-proliferative activity. FK506 and rapamycin share an extraordinarymode of action; they act by recruiting an abundant and ubiquitouslyexpressed cellular protein, the prolyl cis-trans isomerase FKBP, and thebinary complexes subsequently bind to and allosterically inhibit theirtarget proteins calcineurin and mTOR, respectively. Structurally, FK506and rapamycin share a similar FKBP-binding domain but differ in theireffector domains. In FK506 and rapamycin, nature has taught us thatswitching the effector domain of FK506 to that in rapamycin, it ispossible to change the targets from calcineurin to mTOR. The generationof a Rapafucin library of macrocyles that contain FK506 and rapamycinbinding domains should have great potential as new leads for developingdrugs to be used for treating diseases.

With the completion of the sequencing and annotation of the humangenome, a complete catalog of all human proteins encoded in the genomeis now available. The functions of a majority of these proteins,however, remain unknown. One way to elucidate the functions of theseproteins is to find small molecule ligands that specifically bind to theproteins of interest and perturb their biochemical and cellularfunctions. Thus, a major challenge for chemical biologists today is todiscover new small molecule probes for new proteins to facilitate theelucidation of their functions. The recent advance in the development ofprotein chips has offered an exciting new opportunity to simultaneouslyscreen chemical libraries against nearly the entire human proteome. Asingle chip, in the form of a glass slide, is sufficient to display anentire proteome in duplicate arrays. Recently, a protein chip with17,000 human proteins displayed on a single slide has been produced. Amajor advantage of using human protein chips for screening is that theentire displayed proteome can be interrogated at once in a small volumeof assay buffer (<3 mL). Screening of human protein chips, however, isnot yet feasible with most, if not all, existing chemical libraries dueto the lack of a universal readout for detecting the binding of a ligandto a protein on these chips. While it is possible to add artificial tagsto individual compounds in a synthetic library, often the added tagsthemselves interfere with the activity of ligands. Thus, there remains aneed for new compounds and methods for screening chemical librariesagainst the human proteome.

SUMMARY OF THE INVENTION

The present disclosure is directed to a library of Rapafucin compounds,methods of making these compounds, and methods of using the same. Thepresent disclosure is further directed to DNA-encoded libraries ofhybrid cyclic molecules, and more specifically to DNA-encoded librariesof hybrid cyclic compounds based on the immunophilin ligand family ofnatural products FK506 and rapamycyin.

In some embodiments, the Rapafucin compounds in the present disclosurecan have a structure according to Formula (V) or an optically purestereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the groups in Table 1; A is CH₂, NH, NMe, O,S(O)₂ or S; each D is independently O, NMe, or NH; E is CH or N.

Each of R¹, R², R³, and R⁴ can be independently selected from the groupconsisting of H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂,CO₂C₁₋₂₀alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₁₋₁₀alkoxy,C₆₋₁₅arlyl, C₆₋₁₅ayloxy, C₆₋₁₅arylthio, C₂₋₁₀carboxyl, C₁₋₁₀alkylamino,thiol, C₁₋₁₀alkyldisulfide, C₆₋₁₅arylthio, C₁₋₁₀heteroarylthio,(C₃₋₈cycloalkyl)thio, C₂₋₁₀heterocyclylthio, sulfonyl,C₁₋₁₀alkylsulfonyl, amido, C₁₋₁₀alkylamido, selenol, C₁₋₁₀alkylselenol,C₆₋₁₅arylselenol, C₁₋₁₀heteroarylselenol, (C₃₋₈cycloalkyl)selenol,C₂₋₁₀heterocyclylselenol, guanidino, C₁₋₁₀alkylguanidino, urea,C₁₋₁₀alkylurea, ammonium, C₁₋₁₀alkylammonium, cyano, C₁₋₁₀alkylcyano,C₁₋₁₀alkylnitro, adamantine, phosphonate, C₁₋₁₀alkylphosphonate, andC₆₋₁₅arylphosphonate, each of the above can be optionally substitutedwith H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₂₀alkyl, substitutedC₁₋₂₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino,acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl,aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino,aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl,C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substitutedC₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino,(carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl,(C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy,(C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl,C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy,substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substitutedC₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl,C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl.

n is an integer selected from 0 to 4; m is an integer selected from 0 to5; each p is independently an integer selected from 0 to 2; q is aninteger selected from 1 to 10.

Or any R⁴ forms a cyclic structure formed with any R³, the cyclicstructure so formed is selected from the group consisting ofC₂₋₁₀heterocyclyl and C₁₋₁₀heteroaryloptionally substituted with H,halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substitutedC₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino,acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl,aminocarbonyl C₁₋₁₀alkyl aminocarbonylamino, aminodicarbonylamino,aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl,C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substitutedC₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino,(carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl,(C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy,(C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl,C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy,substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substitutedC₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl,C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl.

In some embodiments, q can be 1. In some embodiments, q can be 2. Insome embodiments, q can be 3. In some embodiments, q can be 4. In someembodiments, q can be 5. In some embodiments, q can be 6. In someembodiments, q can be 7. In some embodiments, q can be 8. In someembodiments, q can be 9. In some embodiments, q can be 10. In specificembodiments, q is 3 or 4.

Further provided herein is a macrocyclic compound of Formula (XII) or apharmaceutically acceptable salt, solvate, or stereoisomer thereof:

In some embodiments, Ring A is a 5-10 membered aryl, cycloalkyl,heteroaryl or heterocycloalkyl, optionally substituted with 1-17substituents, each of which is independently selected from the groupconsisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl,haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; J is independently at each occurrence selected from thegroup consisting of —C(O)NR⁶—.

wherein R⁶ is each hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R′ is hydrogen, alkyl,arylalkyl, or haloalkyl; D is independently at each occurrence anoligonucleotide; L^(b) and L^(c) are independently at each occurrenceselected from the group consisting of bond, —O—, —S—, —OC(O)—, —C(O)O—,—(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—,—NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆alkylene (CH₂)_(n)—,optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)nNR⁵C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)OC₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)OC(O)C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆alkylene (CH₂)n-, optionally substituted(CH₂)_(n)—S—C₁₋₆alkylene (CH₂)_(n)—, and optionally substituted(CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or2 groups independently selected from the group consisting of of halo,hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano,nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; X is O, S or NR⁸, wherein R⁸is hydrogen, hydroxy, OR⁹, NR¹⁰, R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ areeach independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionallysubstituted with 1-10 substituents, each of which is selected from thegroup consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano,haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen,hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is eachindependently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl,haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸,CR¹⁸, N, or and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond,—O—, —NR¹⁹—, —SO—, —SO₂—, (CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6membered heteroaryl, optionally substituted with 1-4 substituents, eachof which is independently selected from the group consisting ofhydrogen, hydroxyl, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano,alkylthio, amino, alkylamino, dialkylamino and

wherein each R¹⁹, R²⁰, and R²¹ is independently is selected from thegroup consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula(XIIa):

In some embodiments, each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f),k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b),X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently abond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃)alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ isindependently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ ishydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a),R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f),R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) isindependently selected from the group consisting of hydrogen, halo,amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted alkylamino,optionally substituted dialkylamino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted arylalkyl,optionally substituted heteroarylalkyl and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnaturalamino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

wherein each t is independently an integer selected from 0, 1, 2, 3, 4,5, 6, 7, 8, 9, and 10; R²⁹ is a hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³²are each independently hydrogen or alkyl; X³ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

wherein X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

wherein R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ issubstituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-,—(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; provided that when R is

L is ethylene, X is O, W is

V is

-L⁶-L⁷-L⁸- is

then -L¹-L²-L³-L⁴-L⁵- is not

and; wherein Ring A is substituted with at least one

or at least one of R², R³ R^(2a), R^(3a), R^(2b), R^(3b), R^(2c),R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g),R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring Csubstituted with at least one

or wherein at least one of the linking groups selected from Table 1 issubstituted with at least one

In another aspect, provided herein is a tagged macrocyclic compound of acompound of Formula (XII):

with a compound of Formula (XIV):

Q′-L^(c)-D   Formula (XIV)

Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl orheterocycloalkyl, optionally substituted with 1-17 substituents, each ofwhich is independently selected from the group consisting of hydrogen,hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio,oxo, amino, alkylamino, dialkylamino,

wherein

is a resin;

L^(b) and L^(c) are independently selected from the group consisting ofa bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—,—(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—,optionally substituted (CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionallysubstituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)OC ₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)—S—C₁₋₆alkylene-(CH₂)_(n)—, and optionally substituted(CH₂CH₂₀)_(n); wherein each alkylene is optionally substituted with 1 or2 groups independently selected from the group consisting of of halo,hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano,nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl;

Q and Q′ are independently selected from the group consisting of —N₃,—C≡CH, NR⁶R⁷, —COOH, —ONH₂, —SH, —NH₂,

—(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl,arylalkyl, or haloalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen,hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ areeach independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionallysubstituted with 1-10 substituents, each of which is selected from thegroup consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano,haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen,hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is eachindependently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl,haloalkoxy, aryl, arylalkyl, or heteroaryl;

Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸,CR¹⁸, N, and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond,—O—, —NR¹⁹—, —SO—, —SO₂—, —(CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6membered heteroaryl, optionally substituted with 1-4 substituents, eachof which is independently selected from the group consisting ofhydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano,alkylthio, amino, alkylamino, dialkylamino and

wherein R¹⁹ is selected from the group consisting of hydrogen, hydroxy,OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula(XIIa):

each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i)is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f),X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—,—S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄)alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1f), R^(1i), and R⁴ isindependently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ ishydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a),R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f),R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) isindependently selected from the group consisting of hydrogen, halo,amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted alkylamino,optionally substituted dialkylamino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted arylalkyl,optionally substituted heteroarylalkyl, and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnaturalamino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7,8, 9, and 10; R²⁹ is hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³²are each independently hydrogen or alkyl; X³ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; and provided that when RingA is

L^(a) is ethylene, X is O, W is

V¹ is

V² is

Z is

-L⁶-L⁷-L⁸- is

and -L¹-L²-L³-L⁴-L⁵- is not

D is an oligonucleotide; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c),R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g),R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring Csubstituted with at least one

or wherein at least one of the linking groups selected from Table 1 issubstituted with at least one

DETAILED DESCRIPTION OF THE INVENTION

Nature is a bountiful source of bioactive small molecules that display adizzying array of cellular activities thanks to the evolution processover billions of years. Rapamycin and FK506 comprise a unique structuralfamily of macrocyclic natural products with an extraordinary mode ofaction. On entering cells, both compounds form binary complexes withFKBP12 as well as other members of the FKBP family. The FKBP12—rapamycincomplex can then bind to mTOR and block its kinase activity towardsdownstream substrates such as p70S6K and 4E-BP, while the FKBP12-FK506complex interacts with calcineurin, a protein phosphatase whoseinhibition prevents calcium-dependent signaling and T cell activation.The ability of rapamycin and FK506 to bind FKBPs confers a number ofadvantages for their use as small molecule probes in biology as well asdrugs in medicine. First, the binding of both rapamycin and FK506 toFKBP dramatically increases their effective sizes, allowing forallosteric blockade of substrates to the active sites of mTOR orcalcineurin through indirect disruption of protein-protein interactions.Second, the abundance and ubiquitous expression of intracellular FKBPsserves to enrich rapamycin and FK506 in the intracellular compartmentand maintain their stability. Third, as macrocycles, FK506 and rapamycinare capable of more extensive interactions with proteins than smallermolecules independent of their ability to bind FKBP. Last, but notleast, the high-level expression of FKBPs in blood cells renders themreservoirs and carriers of the drugs for efficient delivery in vivo. Itis thus not surprising that both rapamycin and FK506 became widely useddrugs in their natural forms without further chemical modifications.

Both rapamycin and FK506 can be divided into two structural andfunctional domains: an FKBP-binding domain (FKBD) and an effector domainthat mediates interaction with mTOR or calcineurin, respectively. Thestructures of the FKBDs of rapamycin and FK506 are quite similar, buttheir effector domains are different, accounting for their exclusivetarget specificity. The presence of the separable and modular structuraldomains of FK506 and rapamycin have been extensively exploited togenerate new analogues of both FK506 and rapamycin, including chemicalinducers of dimerization and a large number of rapamycin analogues,known as rapalogs, to alter the specificity of rapamycin for the mutatedFKBP-rapamycin binding domain of mTOR and to improve the toxicity andsolubility profiles of rapamycin. The existence of two distinct FKBDcontaining macrocycles with distinct target specificity also raised theintriguing question of whether replacing the effector domains ofrapamycin or FK506 could further expand the target repertoire of theresultant macrocycles. In their pioneering work, Chakraborty andcolleagues synthesized several rapamycin—peptide hybrid molecules, whichretained high affinity for FKBP but showed no biological activity. Morerecently, we and others independently attempted to explore thispossibility by making larger libraries of the FKBD-containingmacrocycles. In one study, a much larger library of FKBD-containingmacrocycles was made with a synthetic mimic of FKBD, but the resultantmacrocycles suffered from a significant loss in binding affinity forFKBP12, probably accounting for the lack of bioactive compounds fromthat library. Using a natural FKBD extracted from rapamycin, we alsoobserved a significant loss in FKBP binding affinity on formation ofmacrocycles (vide infra).

A Rapafucin library was synthesized as described in WO2017/136708.Rapadocin compound and analogs thereof are disclosed in WO2017/136717,which are used for inhibiting human equilibrative nucleoside transporter1 (ENT1). Rapaglutins and analogs thereof are disclosed inWO2017/136731, which are used as inhibitors of cell proliferation anduseful for the treatment of cancer. Approximately 45,000 compounds weregenerated and ongoing screening of the library as described inWO2018/045250 identified several compounds as being inhibitors of MIFnuclease activity. All of these references are incorporated herein byreference.

In a continuing effort to explore the possibility to using FKBDcontaining macrocycles to target new proteins, we attempted to optimizeand succeeded in identifying FKBDs that allowed for significantretention of binding affinity for FKBP12 upon incorporation intomacrocycles. We also established a facile synthetic route for parallelsynthesis of a large number of FKBD-containing macrocycles.

Below are some acronyms used in the present disclosure. 2-MeTHF refersto 2-methyltetrahydrofuran; DMF refers to dimethylformamide; DMSO refersto dimethyl sulfoxide; DCM refers to dichloromethane; HATU refers to1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate; DIEA refers to N,N-Diisopropylethylamine;TFA refers to trifluoroacetic acid; Fmoc refers tofluorenylmethyloxycarbonyl; MeOH refers to methanol; EtOAc refers toethyl acetate; MgSO₄ refers to magnesium sulfate; COMU-PF₆ refers to(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeniumhexafluorophosphate; CAN refers to acetonitrile; Oxyma refers to ethylcyanohydroxyiminoacetate; LC-MS refers to liquid chromatography-massspectrometry; T3P refers to n-propanephosphonic acid anhydride; SPPSrefers to solid-phase peptide synthesis.

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. The term“comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements. Theterm “about” will be understood by persons of ordinary skill in the art.Whether the term “about” is used explicitly or not, every quantity givenherein refers to the actual given value, and it is also meant to referto the approximation to such given value that would be reasonablyinferred based on the ordinary skill in the art.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology may be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. A person of ordinary skill in the artwould recognize that the above definitions are not intended to includeimpermissible substitution patterns (e.g., methyl substituted with 5different groups, pentavalent carbon, and the like). Such impermissiblesubstitution patterns are easily recognized by a person of ordinaryskill in the art. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. All sequences provided in the disclosed Genbank Accessionnumbers are incorporated herein by reference. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Alkyl groups refer to univalent groups derived from alkanes by removalof a hydrogen atom from any carbon atom, which include straight chainand branched chain with from 1 to 12 carbon atoms, and typically from 1to about 10 carbons or in some embodiments, from 1 to about 6 carbonatoms, or in other embodiments having 1, 2, 3 or 4 carbon atoms.Examples of straight chain alkyl groups include, but are not limited to,methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups. Examplesof branched chain alkyl groups include, but are not limited toisopropyl, isobutyl, sec-butyl and tert-butyl groups. Alkyl groups maybe substituted or unsubstituted. Representative substituted alkyl groupsmay be mono-substituted or substituted more than once, such as, but notlimited to, mono-, di-, or tri-substituted. As used herein, the termalkyl, unless otherwise stated, refers to both cyclic and noncyclicgroups.

The terms “cyclic alkyl” or “cycloalkyl” refer to univalent groupsderived from cycloalkanes by removal of a hydrogen atom from a ringcarbon atom. Cycloalkyl groups are saturated or partially saturatednon-aromatic structures with a single ring or multiple rings includingisolated, fused, bridged, and spiro ring systems, having 3 to 14 carbonatoms, or in some embodiments, from 3 to 12, or 3 to 10, or 3 to 8, or3, 4, 5, 6 or 7 carbon atoms. Cycloalkyl groups may be substituted orunsubstituted. Representative substituted cycloalkyl groups may bemono-substituted or substituted more than once, such as, but not limitedto, mono-, di-, or tri-substituted. Examples of monocyclic cycloalkylgroups include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl groups. Examples of multi-cyclic ringsystems include, but are not limited to, bicycle[4.4.0]decane,bicycle[2.2.1]heptane, spiro[2.2]pentane, and the like. (Cycloalkyl)oxyrefers to —O-cycloalkyl. (Cycloalkyl)thio refers to —S-cycloalkyl. Thisterm also encompasses oxidized forms of sulfur, such as—S(O)-cycloalkyl, or —S(O)₂-cycloalkyl.

Alkenyl groups refer to straight and branched chain and cycloalkenylgroups as defined above, with one or more double bonds between twocarbon atoms. Alkenyl groups may have 2 to about 12 carbon atoms, or insome embodiment from 1 to about 10 carbons or in other embodiments, from1 to about 6 carbon atoms, or 1, 2, 3 or 4 carbon atoms in otherembodiments. Alkenyl groups may be substituted or unsubstituted.Representative substituted alkenyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di-, ortri-substituted. Examples of alkenyl groups include, but are not limitedto, vinyl, allyl, —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂, cyclopentenyl,cyclohexenyl, butadienyl, pentadienyl, and hexadienyl, among others.

Alkynyl groups refer to straight and branched chain and cycloalknylgroups as defined above, with one or more triple bonds between twocarbon atoms. Alkynyl groups may have 2 to about 12 carbon atoms, or insome embodiment from 1 to about 10 carbons or in other embodiments, from1 to about 6 carbon atoms, or 1, 2, 3 or 4 carbon atoms in otherembodiments. Alkynyl groups may be substituted or unsubstituted.Representative substituted alkynyl groups may be mono-substituted orsubstituted more than once, such as, but not limited to, mono-, di-, ortri-substituted. Exemplary alkynyl groups include, but are not limitedto, ethynyl, propargyl, and —C≡C(CH₃), among others.

Aryl groups are cyclic aromatic hydrocarbons that include single andmultiple ring compounds, including multiple ring compounds that containseparate and/or fused aryl groups. Aryl groups may contain from 6 toabout 18 ring carbons, or in some embodiments from 6 to 14 ring carbonsor even 6 to 10 ring carbons in other embodiments. Aryl group alsoincludes heteroaryl groups, which are aromatic ring compounds containing5 or more ring members, one or more ring carbon atoms of which arereplaced with heteroatom such as, but not limited to, N, O, and S. Arylgroups may be substituted or unsubstituted. Representative substitutedaryl groups may be mono-substituted or substituted more than once, suchas, but not limited to, mono-, di-, or tri-substituted. Aryl groupsinclude, but are not limited to, phenyl, biphenylenyl, triphenylenyl,naphthyl, anthryl, and pyrenyl groups. Aryloxy refers to —O-aryl.Arylthio refers to —S-aryl, wherein aryl is as defined herein. This termalso encompasses oxidized forms of sulfur, such as —S(O)-aryl, or—S(O)₂-aryl. Heteroaryloxy refers to —O-heteroaryl. Heteroarylthiorefers to —S-heteroaryl. This term also encompasses oxidized forms ofsulfur, such as —S(O)-heteroaryl, or —S(O)₂-heteoaryl.

Suitable heterocyclyl groups include cyclic groups with atoms of atleast two different elements as members of its rings, of which one ormore is a heteroatom such as, but not limited to, N, O, or S.Heterocyclyl groups may include 3 to about 20 ring members, or 3 to 18in some embodiments, or about 3 to 15, 3 to 12, 3 to 10, or 3 to 6 ringmembers. The ring systems in heterocyclyl groups may be unsaturated,partially saturated, and/or saturated. Heterocyclyl groups may besubstituted or unsubstituted. Representative substituted heterocyclylgroups may be mono-substituted or substituted more than once, such as,but not limited to, mono-, di-, or tri-substituted. Exemplaryheterocyclyl groups include, but are not limited to, pyrrolidinyl,tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl, tetrahydrothiopyranyl,piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl,azetidinyl, aziridinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl,tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl,pyrrolyl, imidazolyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, oxetanyl, thietanyl,homopiperidyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,1,2,3,6-tetrahydropyridyl, indolinyl, 2H-pyranyl, 4H-pyranyl,dioxolanyl, dioxanyl, purinyl, quinolizinyl, cinnolinyl, phthalazinyl,pteridinyl, and benzothiazolyl groups. Heterocyclyloxy refers to—O-heterocycyl. Heterocyclylthio refers to —S-heterocycyl. This termalso encompasses oxidized forms of sulfur, such as —S(O)-heterocyclyl,or —S(O)₂-heterocyclyl.

Polycyclic or polycyclyl groups refer to two or more rings in which twoor more carbons are common to the two adjoining rings, wherein the ringsare “fused rings”; if the rings are joined by one common carbon atom,these are “spiro” ring systems. Rings that are joined throughnon-adjacent atoms are “bridged” rings. Polycyclic groups may besubstituted or unsubstituted. Representative polycyclic groups may besubstituted one or more times.

Halogen groups include F, Cl, Br, and I; nitro group refers to —NO₂;cyano group refers to —CN; isocyano group refers to —N≡C; epoxy groupsencompass structures in which an oxygen atom is directly attached to twoadjacent or non-adjacent carbon atoms of a carbon chain or ring system,which is essentially a cyclic ether structure. An epoxide is a cyclicether with a three-atom ring.

An alkoxy group is a substituted or unsubstituted alkyl group, asdefined above, singular bonded to oxygen. Alkoxy groups may besubstituted or unsubstituted. Representative substituted alkoxy groupsmay be substituted one or more times. Exemplary alkoxy groups include,but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy,hexoxy, isopropoxy, sec-butoxy, tert-butoxy, cyclopropyloxy,cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy groups.

Thiol refers to —SH. Thiocarbonyl refers to (═S). Sulfonyl refers to—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-cycloalkyl, —SO₂-substitutedcycloalkyl, —SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl,—SO₂-substituted heteroaryl, —SO₂-heterocyclyl, and —SO₂-substitutedheterocyclyl. Sulfonylamino refers to —NR^(a)SO₂alkyl,—NR^(a)SO₂-substituted alkyl, —NR^(a)SO₂cycloalkyl,—NR^(a)SO₂substituted cycloalkyl, —NR^(a)SO₂aryl, —NR^(a)SO₂substitutedaryl, —NR^(a)SO₂heteroaryl, —NR^(a)SO₂ substituted heteroaryl,—NR^(a)SO₂heterocyclyl, —NR^(a)SO₂ substituted heterocyclyl, whereineach R^(a) independently is as defined herein.

Carboxyl refers to —COOH or salts thereof. Carboxyester refers to—C(O)O-alkyl, —C(O)O-substituted alkyl, —C(O)O-aryl, —C(O)O-substitutedaryl, —C(O)β-cycloalkyl, —C(O)O-substituted cycloalkyl,—C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclyl,and —C(O)O-substituted heterocyclyl. (Carboxyester)amino refers to—NR^(a)C(O)O-alkyl, —NR^(a)C(O)O-substituted alkyl, —NR^(a)—C(O)O-aryl,—NR^(a)C(O)O-substituted aryl, —NR^(a)C(O)β-cycloalkyl,—NR^(a)C(O)O-substituted cycloalkyl, —NR^(a)C(O)O-heteroaryl,—NR^(a)C(O)O-substituted heteroaryl, —NR^(a)C(O)O-heterocyclyl, and—NR^(a)C(O)O-substituted heterocyclyl, wherein R^(a) is as recitedherein. (Carboxyester)oxy refers to —O—C(O)O-alkyl, —O—C(O)O-substitutedalkyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)β-cycloalkyl,—O—C(O)O-substituted cycloalkyl, —O—C(O)O-heteroaryl,—O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclyl, and—O—C(O)O-substituted heterocyclyl. Oxo refers to (═O).

The terms “amine” and “amino” refer to derivatives of ammonia, whereinone of more hydrogen atoms have been replaced by a substituent whichinclude, but are not limited to alkyl, alkenyl, aryl, and heterocyclylgroups. Carbamate groups refers to —O(C═O)NR¹R², where R¹ and R² areindependently hydrogen, aliphatic groups, aryl groups, or heterocyclylgroups.

Aminocarbonyl refers to —C(O)N(R^(b))₂, wherein each R^(b) independentlyis selected from hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) mayoptionally be joined together with the nitrogen bound thereto to form aheterocyclyl or substituted heterocyclyl group, provided that both R^(b)are not both hydrogen. Aminocarbonylalkyl refers to -alkylC(O)N(R^(b))₂,wherein each R^(b) independently is selected from hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl. Also, each R^(b) may optionally be joinedtogether with the nitrogen bound thereto to form a heterocyclyl orsubstituted heterocyclyl group, provided that both R^(b) are not bothhydrogen. Aminocarbonylamino refers to —NR^(a)C(O)N(R^(b))₂, whereinR^(a) and each R^(b) are as defined herein. Aminodicarbonylamino refersto —NR^(a)C(O)C(O)N(R^(b))₂, wherein R^(a) and each R^(b) are as definedherein. Aminocarbonyloxy refers to —O—C(O)N(R^(b))₂, wherein each R^(b)independently is as defined herein. Aminosulfonyl refers to—SO₂N(R^(b))₂, wherein each R^(b) independently is as defined herein.

Imino refers to —N═R^(c) wherein R^(c) may be selected from hydrogen,aminocarbonylalkyloxy, substituted aminocarbonylalkyloxy,aminocarbonylalkylamino, and substituted aminocarbonylalkylamino.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Twosubstituents may be joined together to form a fused five-, six-, orseven-membered carbocyclic or heterocyclic ring consisting of zero tothree heteroatoms, for example forming methylenedioxy or ethylenedioxy.An optionally substituted group may be unsubstituted (e.g., —CH₂CH₃),fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) orsubstituted at a level anywhere in-between fully substituted andmonosubstituted (e.g., —CH₂CF₃). Where substituents are recited withoutqualification as to substitution, both substituted and unsubstitutedforms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

Pharmaceutically acceptable salts of compounds described herein includeconventional nontoxic salts or quaternary ammonium salts of a compound,e.g., from non-toxic organic or inorganic acids. For example, suchconventional nontoxic salts include those derived from inorganic acidssuch as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric,nitric, and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic,and the like. In other cases, described compounds may contain one ormore acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. These salts can likewise be prepared in situ in theadministration vehicle or the dosage form manufacturing process, or byseparately reacting the purified compound in its free acid form with asuitable base, such as the hydroxide, carbonate or bicarbonate of apharmaceutically acceptable metal cation, with ammonia, or with apharmaceutically acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term “treatment” is used interchangeably herein with the term“therapeutic method” and refers to both 1) therapeutic treatments ormeasures that cure, slow down, lessen symptoms of, and/or haltprogression of a diagnosed pathologic conditions, disease or disorder,and 2) and prophylactic/preventative measures. Those in need oftreatment may include individuals already having a particular medicaldisease or disorder as well as those who may ultimately acquire thedisorder (i.e., those needing preventive measures).

The term “subject” as used herein refers to any individual or patient towhich the subject methods are performed. Generally, the subject ishuman, although as will be appreciated by those in the art, the subjectmay be an animal.

The terms “therapeutically effective amount”, “effective dose”,“therapeutically effective dose”, “effective amount,” or the like referto the amount of a subject compound that will elicit the biological ormedical response in a tissue, system, animal or human that is beingsought by administering said compound. Generally, the response is eitheramelioration of symptoms in a patient or a desired biological outcome.Such amount should be sufficient to inhibit MIF activity.

Also disclosed herein are pharmaceutical compositions includingcompounds with the structures of Formula (I). The term “pharmaceuticallyacceptable carrier” refers to a non-toxic carrier that may beadministered to a patient, together with a compound of this disclosure,and which does not destroy the pharmacological activity thereof.Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins such as human serum albumin,buffer substances such as phosphates, glycine, sorbic acid, potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Pharmaceutically acceptable carriers that may be used in thepharmaceutical compositions of this disclosure include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat and self-emulsifying drug delivery systems (SEDDS) such asα-tocopherol, polyethyleneglycol 1000 succinate, or other similarpolymeric delivery matrices.

In pharmaceutical composition comprising only the compounds describedherein as the active component, methods for administering thesecompositions may additionally comprise the step of administering to thesubject an additional agent or therapy. Such therapies include, but arenot limited to, an anemia therapy, a diabetes therapy, a hypertensiontherapy, a cholesterol therapy, neuropharmacologic drugs, drugsmodulating cardiovascular function, drugs modulating inflammation,immune function, production of blood cells; hormones and antagonists,drugs affecting gastrointestinal function, chemotherapeutics ofmicrobial diseases, and/or chemotherapeutics of neoplastic disease.Other pharmacological therapies can include any other drug or biologicfound in any drug class. For example, other drug classes can compriseallergy/cold/ENT therapies, analgesics, anesthetics,anti-inflammatories, antimicrobials, antivirals, asthma/pulmonarytherapies, cardiovascular therapies, dermatology therapies,endocrine/metabolic therapies, gastrointestinal therapies, cancertherapies, immunology therapies, neurologic therapies, ophthalmictherapies, psychiatric therapies or rheumatologic therapies. Otherexamples of agents or therapies that can be administered with thecompounds described herein include a matrix metalloprotease inhibitor, alipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, acytokine, a growth factor, an immunomodulator, a prostaglandin or ananti-vascular hyperproliferation compound.

The term “therapeutically effective amount” as used herein refers to theamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal, individualor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician, which includes one or more of the following:(1) Preventing the disease; for example, preventing a disease, conditionor disorder in an individual that may be predisposed to the disease,condition or disorder but does not yet experience or display thepathology or symptomatology of the disease, (2) Inhibiting the disease;for example, inhibiting a disease, condition or disorder in anindividual that is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder (i.e., arrestingfurther development of the pathology and/or symptomatology), and (3)Ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual that is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology).

As used herein, the terms “combination,” “combined,” and related termsrefer to the simultaneous or sequential administration of therapeuticagents in accordance with this disclosure. For example, a describedcompound may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present disclosureprovides a single unit dosage form comprising a described compound, anadditional therapeutic agent, and a pharmaceutically acceptable carrier,adjuvant, or vehicle. Two or more agents are typically considered to beadministered “in combination” when a patient or individual issimultaneously exposed to both agents. In many embodiments, two or moreagents are considered to be administered “in combination” when a patientor individual simultaneously shows therapeutically relevant levels ofthe agents in a particular target tissue or sample (e.g., in brain, inserum, etc.).

When the compounds of this disclosure are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to the patient. Alternatively, pharmaceutical orprophylactic compositions according to this disclosure comprise acombination of ivermectin, or any other compound described herein, andanother therapeutic or prophylactic agent. Additional therapeutic agentsthat are normally administered to treat a particular disease orcondition may be referred to as “agents appropriate for the disease, orcondition, being treated.”

The compounds utilized in the compositions and methods of thisdisclosure may also be modified by appending appropriate functionalitiesto enhance selective biological properties. Such modifications are knownin the art and include those, which increase biological penetration intoa given biological system (e.g., blood, lymphatic system, or centralnervous system), increase oral availability, increase solubility toallow administration by injection, alter metabolism and/or alter rate ofexcretion.

According to a preferred embodiment, the compositions of this disclosureare formulated for pharmaceutical administration to a subject orpatient, e.g., a mammal, preferably a human being. Such pharmaceuticalcompositions are used to ameliorate, treat or prevent any of thediseases described herein in a subject.

Agents of the disclosure are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

In some embodiments, the present disclosure provides pharmaceuticallyacceptable compositions comprising a therapeutically effective amount ofone or more of a described compound, formulated together with one ormore pharmaceutically acceptable carriers (additives) and/or diluentsfor use in treating the diseases described herein, including, but notlimited to stroke, ischemia, Alzheimer's, ankylosing spondylitis,arthritis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis,asthma atherosclerosis, Crohn's disease, colitis, dermatitisdiverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome,systemic lupus erythematous, nephritis, ulcerative colitis andParkinson's disease. While it is possible for a described compound to beadministered alone, it is preferable to administer a described compoundas a pharmaceutical formulation (composition) as described herein.Described compounds may be formulated for administration in anyconvenient way for use in human or veterinary medicine, by analogy withother pharmaceuticals.

As described in detail, pharmaceutical compositions of the presentdisclosure may be specially formulated for administration in solid orliquid form, including those adapted for the following: oraladministration, for example, drenches (aqueous or non-aqueous solutionsor suspensions), tablets, e.g., those targeted for buccal, sublingual,and systemic absorption, boluses, powders, granules, pastes forapplication to the tongue; parenteral administration, for example, bysubcutaneous, intramuscular, intravenous or epidural injection as, forexample, a sterile solution or suspension, or sustained-releaseformulation; topical application, for example, as a cream, ointment, ora controlled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream or foam; sublingually; ocularly; transdermally; or nasally,pulmonary and to other mucosal surfaces.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations for use in accordance with the present disclosure includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient, which can be combined with a carrier material, toproduce a single dosage form will vary depending upon the host beingtreated, and the particular mode of administration. The amount of activeingredient that can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound, whichproduces a therapeutic effect. Generally, this amount will range fromabout 1% to about 99% of active ingredient. In some embodiments, thisamount will range from about 5% to about 70%, from about 10% to about50%, or from about 20% to about 40%.

In certain embodiments, a formulation as described herein comprises anexcipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent disclosure. In certain embodiments, an aforementionedformulation renders orally bioavailable a described compound of thepresent disclosure.

Methods of preparing formulations or compositions comprising describedcompounds include a step of bringing into association a compound of thepresent disclosure with the carrier and, optionally, one or moreaccessory ingredients. In general, formulations may be prepared byuniformly and intimately bringing into association a compound of thepresent disclosure with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80, Cremophor RH40, and Cremophor E1) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are natural pharmaceuticallyacceptable oils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such as thosedescribed in Pharmacopeia Helvetica, or a similar alcohol. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

In some cases, in order to prolong the effect of a drug, it may bedesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution, which in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe described compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

The pharmaceutical compositions of this disclosure may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers, which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried cornstarch. When aqueoussuspensions and solutions and propylene glycol are administered orally,the active ingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring and/or coloringagents may be added.

Formulations described herein suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent disclosure as an active ingredient. Compounds described hereinmay also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), an active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;humectants, such as glycerol; disintegrating agents, such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate; solution retarding agents, such asparaffin; absorption accelerators, such as quaternary ammoniumcompounds; wetting agents, such as, for example, cetyl alcohol, glycerolmonostearate, and non-ionic surfactants; absorbents, such as kaolin andbentonite clay; lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

Tablets may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made in asuitable machine in which a mixture of the powdered compound ismoistened with an inert liquid diluent. If a solid carrier is used, thepreparation can be in tablet form, placed in a hard gelatin capsule inpowder or pellet form, or in the form of a troche or lozenge. The amountof solid carrier will vary, e.g., from about 25 to 800 mg, preferablyabout 25 mg to 400 mg. When a liquid carrier is used, the preparationcan be, e.g., in the form of a syrup, emulsion, soft gelatin capsule,sterile injectable liquid such as an ampule or nonaqueous liquidsuspension. Where the composition is in the form of a capsule, anyroutine encapsulation is suitable, for example, using the aforementionedcarriers in a hard gelatin capsule shell.

Tablets and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may alternatively or additionallybe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be formulatedfor rapid release, e.g., freeze-dried. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms for oral administration of compounds of thedisclosure include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Besides inert diluents, oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The pharmaceutical compositions of this disclosure may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisdisclosure with a suitable non-irritating excipient, which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisdisclosure is especially useful when the desired treatment involvesareas or organs readily accessible by topical application. Forapplication topically to the skin, the pharmaceutical composition shouldbe formulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this disclosure include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this disclosure may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-administered transdermalpatches are also included in this disclosure.

The pharmaceutical compositions of this disclosure may be administeredby nasal aerosol or inhalation. Such compositions are prepared accordingto techniques well-known in the art of pharmaceutical formulation andmay be prepared as solutions in saline, employing benzyl alcohol orother suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present disclosure to the body. Dissolvingor dispersing the compound in the proper medium can make such dosageforms. Absorption enhancers can also be used to increase the flux of thecompound across the skin. Either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel can control therate of such flux.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the disclosure, includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Such compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Inclusion ofone or more antibacterial and/orantifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like, may be desirable incertain embodiments. It may alternatively or additionally be desirableto include isotonic agents, such as sugars, sodium chloride, and thelike into the compositions. In addition, prolonged absorption of theinjectable pharmaceutical form may be brought about by the inclusion ofagents, which delay absorption such as aluminum monostearate andgelatin.

In certain embodiments, a described compound or pharmaceuticalpreparation is administered orally. In other embodiments, a describedcompound or pharmaceutical preparation is administered intravenously.Alternative routes of administration include sublingual, intramuscular,and transdermal administrations.

When compounds described herein are administered as pharmaceuticals, tohumans and animals, they can be given per se or as a pharmaceuticalcomposition containing, for example, 0.1% to 99.5% (more preferably,0.5% to 90%) of active ingredient in combination with a pharmaceuticallyacceptable carrier.

Preparations described herein may be given orally, parenterally,topically, or rectally. They are of course given in forms suitable forthe relevant administration route. For example, they are administered intablets or capsule form, by injection, inhalation, eye lotion, ointment,suppository, etc. administration by injection, infusion or inhalation;topical by lotion or ointment; and rectal by suppositories. Oraladministrations are preferred.

Such compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, compounds describedherein which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the present disclosure, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the disclosure may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The terms “administration of” and or “administering” should beunderstood to mean providing a pharmaceutical composition in atherapeutically effective amount to the subject in need of treatment.Administration routes can be enteral, topical or parenteral. As such,administration routes include but are not limited to intracutaneous,subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, transdermal,transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid,intraspinal and intrastemal , oral, sublingual buccal, rectal, vaginal,nasal ocular administrations, as well infusion, inhalation, andnebulization.

The crystal structures of the FKBP-FK506-calcineurin andFKBP-rapamycin-TOR complexes revealed that both FK506 and rapamycin canbe divided into two functional domains, the “FKBP-binding domain” (FKBD)and the “effector” domain, which mediate their interactions withcalcineurin and TOR, respectively. While there are extensiveprotein-protein interactions between FKBP and calcinerin in theirternary complex, there are far fewer interactions between FKBP and TOR,suggesting that the key role of FKBP in the inhibition of TOR byrapamycin is to bind to FKBD of the drug and present its effector domainto TOR.

A comparison of the structures of FK506 and rapamycin reveal that theyshare a nearly identical FKBD but each possesses a distinct effectordomain. By swapping the effector domain of FK506 with that of rapamycin,it is possible to change the target from calcineurin to TOR, which bearsno sequence, functional or structural similarities to each other. Inaddition, other proteins may be targeted by grafting new structures ontothe FKBD of FK506 and rapamycin. Thus, the generation of new compoundswith new target specificity may be achieved by grafting a sufficientlylarge combinatorial library onto FKBD in conjunction with proteome-widescreens through which each compound in the library is tested againstevery protein in the human proteome.

In some embodiments, provided herein is a macrocyclic compound accordingto Formula (I), which includes an FKBD, an effector domain, a firstlinker, and a second linker, wherein the FKBD, the effector domain, thefirst linker, and the second linker together form a macrocycle.

In some embodiments, provided herein is a macrocyclic compound accordingto Formula (II) or an optically pure stereoisomer or pharmaceuticallyacceptable salt thereof.

B can be CH₂, NH, NMe, O, S, or S(O)₂; X can be O, NH or NMe; E can beCH or N; n is an integer selected from 0 to 4; m is an integer selectedfrom 1 to 10. AA in this formula represents natural and unnatural aminoacids, each of which can be selected from Table 4 below.

In some embodiments, m can be 1. In some embodiments, m can be 2. Insome embodiments, m can be 3. In some embodiments, m can be 4. In someembodiments, m can be 5. In some embodiments, m can be 6. In someembodiments, m can be 7. In some embodiments, m can be 8. In someembodiments, m can be 9. In some embodiments, m can be 10. In specificembodiment, m is 3 or 4.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl,C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, andCO₂C₁₋₂₀alkyl. R² is selected from the group consisting of C₆₋₁₅aryl andC₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃,NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy,substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy,amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl,aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy,aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy,substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio,carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano,C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy,substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted(C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl,C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio,substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substitutedheterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl.

V is

Z is a bond,

wherein R³ and R⁴ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁵,CR⁵, N, and NR⁵, wherein R⁵ is hydrogen or alkyl.

Each of L¹, L², or L³ can be selected from the group consisting of thestructures shown in Table 1 below.

TABLE 1 The linker structures. optionally substituted optionallysubstituted optionally substituted optionally substituted —(CH₂)_(n)C₁₋₆alkylene —(CH₂)_(n)C₂₋₆ alkenylene —(CH₂)_(n)C₃₋₆ cycloalkylene—(CH₂)_(n)C₃₋₆ cycloalkenylene optionally substituted optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)OC₁₋₆ alkylene —(CH₂)_(n)OC₂₋₆ —(CH₂)_(n)OC₃₋₆ cycloalkylene—(CH₂)_(n)OC₃₋₆ alkenylene cycloalkenylene optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)C(O)C₁₋₆ —(CH₂)_(n)C(O)C₂₋₆ —(CH₂)_(n)C(O)C₃₋₆—(CH₂)_(n)C(O)C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenyleneoptionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)C(O)OC₁₋₆ —(CH₂)_(n)C(O)OC₂₋₆—(CH₂)_(n)C(O)O— —(CH₂)_(n)C(O)OC₃₋₆ alkylene alkenylene C₃₋₆cycloalkylene cycloalkenylene optionally substituted optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)OC(O)C₁₋₆ —(CH₂)_(n)OC(O)C₂₋₆ —(CH₂)_(n)OC(O)—C₃₋₆—(CH₂)_(n)OC(O)C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenyleneoptionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)NR²⁰C₁₋₆ —(CH₂)_(n)NR²⁰C₂₋₆—(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ alkylene alkenylene cycloalkylenecycloalkenylene optionally substituted optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)NR²⁰C(O)C₁₋₆—(CH₂)_(n)NR²⁰C(O)C₂₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆alkylene alkenylene cycloalkylene cycloalkenylene optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)C(O)NR²⁰C₁₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆—(CH₂)_(n)C(O)NR²⁰—C₃₋₆ alkylene alkenylene cycloalkylenecycloalkenylene optionally substituted optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)—S—C₁₋₆ —(CH₂)_(n)—S—C₂₋₆—(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₃₋₆ alkylene alkenylene cycloalkylenecycloalkenylene optionally substituted optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— C₁₋₆ alkylene C₂₋₆ alkenylene C₃₋₆cycloalkylene C₃₋₆ cycloalkenylene optionally substituted optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂—C₂₋₆ —(CH₂)_(n)—SO₂—C₃₋₆—(CH₂)_(n)—SO₂—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenyleneoptionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂— SO₂— C₃₋₆cycloalkylene C₃₋₆ cycloalkenylene C₁₋₆ alkylene C₂₋₆ alkenyleneoptionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)—SO—C₁₋₆ —(CH₂)_(n)—SO—C₂₋₆—(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ alkylene alkenylene cycloalkylenecycloalkenylene optionally substituted optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)——(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO——(CH₂)_(n)C(O)(CH₂)_(n)—SO— SO— SO— C₃₋₆ cycloalkenylene C₃₋₆cycloalkenylene C₁₋₆ alkylene C₂₋₆ alkenylene optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)—S—S—C₁₋₆ —(CH₂)_(n)—S—S—C₂₋₆ —(CH₂)_(n)—S—S—C₃₋₆—(CH₂)_(n)—S—S—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenyleneoptionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S——(CH₂)_(n)C(O)(CH₂)_(n)—S—S— S—C₁₋₆ alkylene S—C₂₋₆ alkenylene C₃₋₆cycloalkylene C₃₋₆ cycloalkenylene optionally substituted optionallysubstituted optionally substituted optionally substituted —(CH₂)_(n)C₁₋₆alkylene- —(CH₂)_(n)C₂₋₆ —(CH₂)_(n)C₃₋₆ cycloalkylene- —(CH₂)_(n)C₃₋₆NR²¹- alkenylene-NR²¹- NR²¹- cycloalkenylene-NR²¹- optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)OC₁₋₆ alkylene- —(CH₂)_(n)OC₂₋₆ —(CH₂)_(n)OC₃₋₆cycloalkylene- —(CH₂)_(n)OC₃₋₆ NR²¹- alkenylene-NR²¹- NR²¹-cycloalkenylene-NR²¹- optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)C₁₋₆—(CH₂)_(n)C(O)C₂₋₆ —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆ alkylene-NR²¹-alkenylene-NR²¹- cycloalkylene-NR²¹- cycloalkenylene-NR²¹- optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)C(O)OC₁₋₆ —(CH₂)_(n)C(O)OC₂₋₆ —(CH₂)_(n)C(O)O—C₃₋₆—(CH₂)_(n)C(O)OC₃₋₆ alkylene-NR²¹- alkenylene-NR²¹- cycloalkylene-NR²¹-cycloalkenylene-NR²¹- optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)OC(O)C₁₋₆—(CH₂)_(n)OC(O)C₂₋₆ —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₃₋₆alkylene-NR²¹- alkenylene-NR²¹- cycloalkylene-NR²¹-cycloalkenylene-NR²¹- optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₁₋₆—(CH₂)_(n)NR²⁰C₂₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ alkylene-NR²¹-alkenylene-NR²¹- cycloalkylene-NR²¹- cycloalkenylene-NR²¹- optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)NR²⁰C(O)C₁₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆—(CH₂)_(n)NR²⁰C(O)— —(CH₂)_(n)NR²⁰C(O)— alkylene-NR²¹- alkenylene-NR²¹-C₃₋₆ cycloalkylene-NR²¹- C₃₋₆ cycloalkenylene-NR²¹- optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)C(O)NR²⁰C₁₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆—(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ alkylene-NR²¹-alkenylene-NR²¹- cycloalkylene-NR²¹- cycloalkenylene-NR²¹- optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)—S—C₁₋₆ —(CH₂)_(n)—S—C₂₋₆ —(CH₂)_(n)—S—C₃₋₆—(CH₂)_(n)—S—C₃₋₆ alkylene-NR²¹- alkenylene cycloalkylene-NR²¹-cycloalkenylene-NR²¹- optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— C₁₋₆ alkylene-NR²¹- C₂₋₆ alkenylene-NR²¹-C₃₋₆ cycloalkylene-NR²¹- C₃₋₆ cycloalkenylene-NR²¹- optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂——(CH₂)_(n)—SO₂—C₃₋₆ alkylene-NR²¹- alkenylene-NR²¹- C₃₋₆cycloalkylene-NR²¹- cycloalkenylene-NR²¹- optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)——(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂— SO₂— C₃₋₆cycloalkylene-NR²¹- C₃₋₆ cycloalkenylene-NR²¹- C₁₋₆ alkylene-NR²¹- C₂₋₆alkenylene-NR²¹- optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—SO—C₁₋₆—(CH₂)_(n)—SO—C₂₋₆ —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ alkylene-NR²¹-alkenylene-NR²¹- cycloalkylene-NR²¹- cycloalkenylene-NR²¹- optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)——(CH₂)_(n)C(O)(CH₂)_(n)—SO— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— SO—C₁₋₆alkylene-NR²¹- SO—C₂₋₆ alkenylene-NR²¹- C₃₋₆ cycloalkylene-NR²¹- C₃₋₆cycloalkenylene-NR²¹- optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—S—S—C₁₋₆—(CH₂)_(n)—S—S—C₂₋₆ —(CH₂)_(n)—S—S— —(CH₂)_(n)—S—S—C₃₋₆ alkylene-NR²¹-alkenylene-NR²¹- C₃₋₆ cycloalkylene-NR²¹- cycloalkenylene-NR²¹-optionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S——(CH₂)_(n)C(O)(CH₂)_(n)—S—S— S—C₁₋₆ alkylene-NR²¹- S—C₂₋₆alkenylene-NR²¹- C₃₋₆ cycloalkylene-NR²¹- C₃₋₆ cycloalkenylene-NR²¹-optionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)C₁₋₆ —(CH₂)_(n)C₂₋₆ —(CH₂)_(n)C₃₋₆—(CH₂)_(n)C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)——cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)OC₁₋₆—(CH₂)_(n)OC₂₋₆ —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₃₋₆ alkylene-C(O)—alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)C(O)C₁₋₆ —(CH₂)_(n)C(O)C₂₋₆ —(CH₂)_(n)C(O)C₃₋₆—(CH₂)_(n)C(O)C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)—cycloalkenylene-C(O)—— optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)OC₁₋₆—(CH₂)_(n)C(O)OC₂₋₆ —(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₃₋₆alkylene-(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)—optionally substituted optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)OC(O)C₁₋₆ —(CH₂)_(n)OC(O)C₂₋₆—(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₃₋₆ alkylene-C(O)— alkenylene-C(O)—cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)NR²⁰C₁₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ —(CH₂)_(n)NR²⁰C₃₋₆—(CH₂)_(n)NR²⁰C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)—cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)C₁₋₆—(CH₂)_(n)NR²⁰C(O)C₂₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)—cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₁₋₆—(CH₂)_(n)NR²⁰C₂₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ alkylene-C(O)—alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)C(O)NR²⁰C₁₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆—(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ alkylene-C(O)—alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —(CH₂)_(n)—S—C₁₋₆ —(CH₂)_(n)—S—C₂₋₆ —(CH₂)_(n)—S—C₃₋₆—(CH₂)_(n)—S—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)—cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— C₁₋₆ alkylene-C(O) C₂₋₆ alkenylene-C(O) C₃₋₆cycloalkylene-C(O) C₃₋₆ cycloalkenylene-C(O) optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂—C₂₋₆ —(CH₂)_(n)—SO₂—C₃₋₆—(CH₂)_(n)—SO₂—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)—cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)——(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂——(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂—C₁₋₆ alkylene-C(O) C₂₋₆ alkenylene-C(O)C₃₋₆ cycloalkylene-C(O) C₃₋₆ cycloalkenylene-C(O) optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)—SO—C₁₋₆ —(CH₂)_(n)—SO—C₂₋₆ —(CH₂)_(n)—SO—C₃₋₆—(CH₂)_(n)—SO—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)—cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)——(CH₂)_(n)C(O)(CH₂)_(n)—SO— —(CH₂)_(n)—C(O)(CH₂)_(n)—SO——(CH₂)_(n)—C(O)(CH₂)_(n)—SO— SO—C₁₋₆ alkylene-C(O) C₂₋₆ alkenylene-C(O)C₃₋₆ cycloalkylene-C(O) C₃₋₆ cycloalkenylene-C(O) optionally substitutedoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)—S—S—C₁₋₆ —(CH₂)_(n)—S—S—C₂₋₆ —(CH₂)_(n)—S—S—C₃₋₆—(CH₂)_(n)—S—S—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)—cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—S—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S——(CH₂)_(n)C(O)(CH₂)_(n)—S—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— C₁₋₆alkylene-C(O) C₂₋₆ alkenylene-C(O) C₃₋₆ cycloalkylene-C(O) C₃₋₆cycloalkenylene-C(O)— optionally substituted optionally substitutedoptionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)OC₁₋₆—NR²⁰C(O)(CH₂)_(n)O—C₂₋₆ —NR²⁰C(O)(CH₂)_(n)O—C₃₋₆—NR²⁰C(O)(CH₂)_(n)O—C₃₋₆ alkylene-(CO) alkenylene-(CO)cycloalkylene-(CO) cycloalkenylene-(CO) optionally substitutedoptionally substituted optionally substituted optionally substituted—NR²⁰C(O)(CH₂)_(n)—S— NR²⁰C(O)(CH₂)_(n)—S— —NR²⁰C(O)(CH₂)_(n)—S——NR²⁰C(O)(CH₂)_(n)—S— C₁₋₆ alkylene-(CO) C₂₋₆ alkenylene-(CO) C₃₋₆cycloalkylene-(CO) C₃₋₆ cycloalkenylene-(CO) optionally substitutedoptionally substituted optionally substituted optionally substituted—NR²⁰C(O)(CH₂)_(n)NR²¹ —NR²⁰C(O)(CH₂)_(n)NR²¹- —NR²⁰C(O)(CH₂)_(n)NR²¹-—NR²⁰C(O)(CH₂)_(n)NR²¹- C₁₋₆ alkylene-(CO) C₂₋₆ alkenylene-(CO) C₃₋₆cycloalkylene-(CO) C₃₋₆ cycloalkenylene-(CO) optionally substitutedoptionally substituted optionally substituted optionally substitutedC(O)NR²⁰(CH₂)_(n)OC₁₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₂₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₃₋₆—C(O)NR²⁰(CH₂)_(n)O—C₃₋₆ alkylene-(CO) alkenylene-(CO)cycloalkylene-(CO) cycloalkenylene-(CO) optionally substitutedoptionally substituted optionally substituted optionally substituted—C(O)NR²⁰(CH₂)_(n)—S— —C(O)NR²⁰(CH₂)_(n)—S— —C(O)NR²⁰(CH₂)_(n)—S——C(O)NR²⁰(CH₂)_(n)—S— C₁₋₆ alkylene-(CO) C₂₋₆ alkenylene-(CO) C₃₋₆cycloalkylene-(CO) C₃₋₆ cycloalkenylene-(CO) optionally substitutedoptionally substituted optionally substituted optionally substituted—C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)— vC(O)NR²⁰(CH₂)_(n)——C(O)NR²⁰(CH₂)_(n)— NR²¹C₁₋₆ alkylene-(CO) NR²¹C₂₋₆ alkenylene-(CO)NR²¹C₃₋₆ cycloalkylene-(CO) NR²¹C₃₋₆ cycloalkenylene-(CO) optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)(CH₂)_(n)C₁₋₆ alkylene —C(O)(CH₂)_(n)C₁₋₆ alkenylene—C(O)(CH₂)_(n)C₃₋₆ cycloalkylene —C(O)(CH₂)_(n)C₃₋₆ —(CH₂)_(n)——(CH₂)_(n)— —(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆—C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)— alkenylene-(CH₂)_(n)—cycloalkylene (CH₂)_(n)— cycloalkenylene (CH₂)_(n)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆—C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O—cycloalkylene (CH₂)_(n)—O— cycloalkenylene (CH₂)_(n)—O— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆—C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O—cycloalkylene (CH₂)_(n)—O— cycloalkenylene (CH₂)_(n)—O— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)(CH₂)_(n)C₁₋₆ —C(O)(CH₂)_(n)C₁₋₆ —C(O)(CH₂)_(n)C₃₋₆—C(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)—cycloalkylene (CH₂)_(n)—C(O)— cycloalkenylene (CH₂)_(n)—C(O)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆—C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)—cycloalkylene (CH₂)_(n)—C(O)— cycloalkenylene (CH₂)_(n)—C(O)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₃₋₆—OC(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)— alkenylene-(CH₂)_(n)—cycloalkylene-(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₃₋₆—O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)— alkenylene-(CH₂)_(n)—cycloalkylene-(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₃₋₆—OC(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O—cycloalkylene-(CH₂)_(n)—O— cycloalkenylene-(CH₂)_(n)—O— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₃₋₆—O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O—cycloalkylene-(CH₂)_(n)—O— cycloalkenylene-(CH₂)_(n)—O— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₃ ₆—OC(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)—cycloalkylene-(CH₂)_(n)—C(O)— cycloalkenylene-(CH₂)_(n)—C(O)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₃₋₆—O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)—cycloalkylene-(CH₂)_(n)—C(O)— cycloalkenylene-(CH₂)_(n)—C(O)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)——C(O)NR²⁰(CH₂)_(n)C₃₋₆ C₁₋₆ alkylene-(CH₂)_(n)— C₁₋₆alkenylene-(CH₂)_(n)— C₃₋₆ cycloalkylene-(CH₂)_(n)—cycloalkenylene-(CH₂)_(n)— optionally substituted optionally substitutedoptionally substituted optionally substituted NR²⁰C(O)(CH₂)_(n)——NR²⁰C(O)(CH₂)_(n)— —NR²⁰C(O)(CH₂)_(n)— —NR²⁰C(O)(CH₂)_(n)C₃₋₆ C₁₋₆alkylene-(CH₂)_(n)— C₁₋₆ alkenylene-(CH₂)_(n)— C₃₋₆cycloalkylene-(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)NR²⁰(CH₂)_(n) —C(O)NR²⁰(CH₂)_(n) —C(O)NR²⁰(CH₂)_(n)——C(O)NR²⁰(CH₂)_(n)C₃₋₆ C₁₋₆ alkylene-(CH₂)_(n)—O— C₁₋₆alkenylene-(CH₂)_(n)—O— C₃₋₆ cycloalkylene-(CH₂)_(n)—O—cycloalkenylene-(CH₂)_(n)—O— optionally substituted optionallysubstituted optionally substituted optionally substituted—NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)——NR²⁰C(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O—C₃₋₆ cycloalkylene-(CH₂)_(n)—O— cycloalkenylene-(CH₂)_(n)—O— optionallysubstituted optionally substituted optionally substituted optionallysubstituted —C(O)NR²⁰(CH₂)_(n)C₁₋₆ —C(O)NR²⁰(CH₂)_(n)C₁₋₆—C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ alkylene-(CH₂)_(n)—C(O)—alkenylene-(CH₂)_(n)—C(O)— C₃₋₆ cycloalkylene-(CH₂)_(n)—C(O)—cycloalkenylene-(CH₂)_(n)—C(O)— optionally substituted optionallysubstituted optionally substituted optionally substituted—NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)——NR²⁰C(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)—alkenylene-(CH₂)_(n)—C(O)— C₃₋₆ cycloalkylene-(CH₂)_(n)—C(O)—cycloalkenylene-(CH₂)_(n)—C(O)— optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₃₋₆—(CH₂)_(n)C₃₋₆ alkynylene heterocycloalkylene heterocycloalkenyleneoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₂₋₆ heterocycloalkyleneheterocycloalkenylene alkynylene optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆—(CH₂)_(n)C(O)C₂₋₆ heterocycloalkylene heterocycloalkenylene alkynyleneoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)C(O)OC₃₋₆ —(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₂₋₆heterocycloalkylene heterocycloalkenylene alkynylene optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)OC(O)C₃₋₆ —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₂₋₆heterocycloalkylene heterocycloalkenylene alkynylene optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰—C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆heterocycloalkylene heterocycloalkenylene alkynylene optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆heterocycloalkylene heterocycloalkenylene alkynylene optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)C(O)NR²⁰- —(CH₂)_(n)C(O)NR²⁰- —(CH₂)_(n)C(O)NR²⁰- optionallysubstituted optionally substituted C₂₋₆ alkynylene C₃₋₆heterocycloalkylene C₃₋₆ heterocycloalkenylene optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—S——(CH₂)_(n)—S— —(CH₂)_(n)—S—C₂₋₆ C₃₋₆ C₃₋₆ alkynylene heterocycloalkyleneheterocycloalkenylene optionally substituted optionally substitutedoptionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— C₃₋₆heterocycloalkylene C₃₋₆ heterocycloalkenylene C₂₋₆ alkynyleneoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂— —(CH₂)_(n)—SO₂—C₂₋₆heterocycloalkylene C₃₋₆ heterocycloalkenylene alkynylene optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)——(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂— SO₂— C₂₋₆ alkynylene C₃₋₆heterocycloalkylene C₃₋₆ heterocycloalkenylene optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—SO—C₃₋₆—(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₂₋₆ heterocycloalkyleneheterocycloalkenylene alkynylene optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)——(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— SO— SO— C₂₋₆alkynylene C₃₋₆ heterocycloalkylene C₃₋₆ heterocycloalkenyleneoptionally substituted optionally substituted optionally substituted—(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₂₋₆heterocycloalkylene heterocycloalkenylene alkynylene optionallysubstituted optionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S——(CH₂)_(n)C(O)(CH₂)_(n)—S—S— S— S— C₂₋₆ alkynylene C₃₋₆heterocycloalkylene C₃₋₆ heterocycloalkenylene optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C₃₋₆—(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)OC₃₋₆—(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)₃₋₆—(CH₂)_(n)C(O)—C₃₋₆ —(CH₂)_(n)C(O)C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)O—C₃₋₆—(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)OC(O)—C₃₋₆—(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₃₋₆—(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)—C₃₋₆—(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰—C₃₋₆—(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—S—C₃₋₆—(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene optionally substituted optionallysubstituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₂₋₆heterocycloalkylene-NR²¹- heterocycloalkenylene-NR²¹- alkynylene-NR²¹-optionally substituted optionally substituted optionally substituted—(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂—C₁₋₆heterocycloalkylene-NR²¹- heterocycloalkenylene-NR²¹- alkynylene-NR²¹-optionally substituted optionally substituted optionally substituted—(CH₂)_(n)—C(O)(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—SO—C₃₋₆—(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—S—S—C₃₋₆—(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-NR²¹-heterocycloalkenylene-NR²¹- alkynylene-NR²¹- optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C₃₋₆—(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)OC₃₋₆—(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)C₃₋₆—(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)OC₃₋₆—(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)OC(O)C₃₋₆—(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₃₋₆—(CH₂)_(n)NR²⁰—C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ heteroalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)C₃₋₆—(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₃₋₆—(CH₂)_(n)NR²⁰—C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰C₃₋₆—(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—S—C₃₋₆—(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-C(O)heterocycloalkenylene-C(O) alkynylene-C(O) optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)SO₂—C₃₋₆—(CH₂)_(n)SO₂—C₃₋₆ —(CH₂)_(n)SO₂—C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₂₋₆ heterocycloalkylene-C(O)heterocycloalkenylene-C(O) alkynylene-C(O) optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—SO—C₃₋₆—(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-C(O)heterocycloalkenylene-C(O) alkynylene-C(O) optionally substitutedoptionally substituted optionally substituted —(CH₂)_(n)—S—S—C₃₋₆—(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-C(O)—heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substitutedoptionally substituted optionally substituted—(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆—(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-C(O)heterocycloalkenylene-C(O) alkynylene-C(O) optionally substitutedoptionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)O—C₃₋₆—NR²⁰C(O)(CH₂)_(n)O—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)O—C₂₋₆heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O)optionally substituted optionally substituted optionally substitutedNR²⁰C(O)(CH₂)_(n)—S—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—S—C₃₋₆—NR²⁰C(O)(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-C(O)heterocycloalkenylene-C(O) alkynylene-C(O) optionally substitutedoptionally substituted optionally substituted—NR²⁰C(O)(CH₂)_(n)NR²¹—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)NR²¹—C₃₋₆—NR²⁰C(O)(CH₂)_(n)NR²¹—C₂₋₆ heterocycloalkylene-C(O)heterocycloalkenylene-C(O) alkynylene-C(O) optionally substitutedoptionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)O—C₃₋₆—C(O)NR²⁰(CH₂)_(n)O—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₂₋₆heterocycloalkylene-(CO) heterocycloalkenylene-(CO) alkynylene-(CO)optionally substituted optionally substituted optionally substituted—C(O)NR²⁰(CH₂)_(n)—S—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—S—C₃₋₆—C(O)NR²⁰(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-(CO)heterocycloalkenylene-(CO) alkynylene-(CO) optionally substitutedoptionally substituted optionally substituted—C(O)NR²⁰(CH₂)_(n)—NR²¹—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—NR²¹—C₃₋₆—C(O)NR²⁰(CH₂)_(n)—NR²¹C₂₋₆ heterocycloalkylene-(CO)heterocycloalkenylene-(CO) alkynylene-(CO) optionally substitutedoptionally substituted optionally substituted —C(O)(CH₂)_(n)C₃₋₆—C(O)(CH₂)_(n)C₃₋₆ —C(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene-(CH₂)_(n)—heterocycloalkenylene-(CH₂)_(n)— alkynylene-(CH₂)_(n)— optionallysubstituted optionally substituted optionally substituted—C(O)O(CH₂)_(n)—C₃₋₆ —C(O)O(CH₂)_(n)—C₃₋₆ —C(O)O(CH₂)_(n)C₁₋₆heterocycloalkylene-(CH₂)_(n)— heterocycloalkenylene-(CH₂)_(n)—alkynylene-(CH₂)_(n)— optionally substituted optionally substitutedoptionally substituted —C(O)(CH₂)_(n)—C₃₋₆ —C(O)(CH₂)_(n)—C₃₋₆—C(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene-alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O— optionally substitutedoptionally substituted optionally substituted —C(O)O(CH₂)_(n)—C₃₋₆—C(O)O(CH₂)_(n)—C₃₋₆ —C(O)O(CH₂)_(n)C₁₋₆ heterocycloalkylene-heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O—optionally substituted optionally substituted optionally substituted—C(O)(CH₂)_(n)C₃₋₆ —C(O)(CH₂)_(n)—C₃₋₆ —C(O)(CH₂)_(n)C₁₋₆heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)—(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionally substituted optionallysubstituted optionally substituted —C(O)(CH₂)_(n)C₃₋₆—C(O)(CH₂)_(n)—C₃₋₆ —C(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene-heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)—(CH₂)_(n)—C(O)— optionally substituted optionally substituted optionallysubstituted —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)—C₃₋₆ —OC(O)(CH₂)_(n)C₁₋₆heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)(CH₂)_(n) (CH₂)_(n) optionally substituted optionally substitutedoptionally substituted —O(CH₂)_(n)—C₃₋₆ —O(CH₂)_(n)—C₃₋₆—O(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene-alkynylene-(CH₂)_(n) (CH₂)_(n) (CH₂)_(n) optionally substitutedoptionally substituted optionally substituted —OC(O)(CH₂)_(n)C₃₋₆—OC(O)(CH₂)_(n)—C₃₋₆ —OC(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene-heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O—optionally substituted optionally substituted optionally substituted—O(CH₂)_(n)C₃₋₆ —O(CH₂)_(n)C₃₋₆ —O(CH₂)_(n)C₁₋₆ heterocycloalkylene-heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O—optionally substituted optionally substituted optionally substituted—OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)C₁₋₆heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)—(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionally substituted optionallysubstituted optionally substituted —O(CH₂)_(n)—C₃₋₆ —O(CH₂)_(n)—C₃₋₆—O(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene-alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionallysubstituted optionally substituted optionally substituted—C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₁₋₆heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—(CH₂)_(n)— (CH₂)_(n)— optionally substituted optionally substitutedoptionally substituted —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₃₋₆NR²⁰C(O)(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene-alkynylene-(CH₂)_(n)— (CH₂)_(n)— (CH₂)_(n)— optionally substitutedoptionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)—C₃₋₆—C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₁₋₆ heterocycloalkylene-heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O—optionally substituted optionally substituted optionally substituted—NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₁₋₆heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—O—(CH₂)_(n)—O— (CH₂)_(n)—O— optionally substituted optionally substitutedoptionally substituted —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₃₋₆—C(O)NR²⁰(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene-alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionallysubstituted optionally substituted optionally substituted—NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₁₋₆heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)—(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)—* Each R²⁰ and R²¹ is independently selected from the group consistingof hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl.

In some embodiments, the FKBD-containing moiety before incorporated intothe macrocycle can have a structure according to Formula (III) or anoptically pure stereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the structure in Table 1; A is CH₂, NH, O, orS; each X is independently O, NH, or NMe; E is CH or N;

represents a single or a double bond. n is an integer selected from 0 to4.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl,C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, andCO₂C₁₋₂₀alkyl. R² is selected from the group consisting of H, halogen,hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, andCO₂C₁₋₂₀alkyl. R³ is selected from the group consisting of C₆₋₁₅aryl andC₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃,NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy,substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy,amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl,aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy,aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy,substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio,carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano,C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy,substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted(C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl,C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio,substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substitutedheterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl.

V is

Z is a bond,

wherein R⁴ and R⁵ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁶,CR⁶, N, and NR⁶, wherein R⁶ is hydrogen or alkyl.

In some embodiments, the FKBD-containing moiety before incorporated intothe macrocycle can have a structure according to Formula (IV) or anoptically pure stereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the structures in Table 1; A is CH₂, NH, O,or S; each X is independently O or NH; E is CH or N; each R¹ is selectedfrom the group consisting of H, halogen, hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂,NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, and CO₂C₁₋₂₀alkyl; each R² isselected from the group consisting of H, halogen, hydroxyl, N₃, NH₂,NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substitutedC₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino,substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl,aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy,aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy,substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio,carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano,C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy,substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted(C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl,C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio,substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substitutedheterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl; n is an integer selected from 0 to 4;and m is an integer selected from 0 to 5.

In some embodiments, the Rapafucin compounds in the present disclosurecan have a structure according to Formula (V) or an optically purestereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the groups in Table 1 ; A is CH₂, NH, NMe, O,S(O)₂ or S; each X is independently O, NMe, or NH; E is CH or N.

Each of R¹, R², R³, and R⁴ can be independently selected from the groupconsisting of H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂,COC₁₋₂₀alkyl, CO₂C₁₋₂₀alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₁₋₁₀alkoxy, C₆₋₁₅aryl, C₆₋₁₅aryloxy, C₆₋₁₅arylthio, C₂₋₁₀carboxyl,C₁₋₁₀alkylamino, thiol, C₁₋₁₀alkyldisulfide, C₆₋₁₅arylthio,C₁₋₁₀heteroarylthio, (C₃₋₈cycloalkyl)thio, C₂₋₁₀heterocyclylthio,sulfonyl, C₁₋₁₀alkylsulfonyl, amido, C₁₋₁₀alkylamido, selenol,C₁₋₁₀alkylselenol, C₆₋₁₅arylselenol, C₁₋₁₀heteroarylselenol,(C₃₋₈cycloalkyl)selenol, C₂₋₁₀heterocyclylselenol, guanidino,C₁₋₁₀alkylguanidino, urea, C₁₋₁₀alkylurea, ammonium, C₁₋₁₀alkylammonium,cyano, C₁₋₁₀alkylcyano, C₁₋₁₀alkylnitro, adamantine, phosphonate,C₁₋₁₀alkylphosphonate, and C₆₋₁₅arylphosphonate, each of the above canbe optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃,C₁₋₂₀alkyl, substituted C₁₋₂₀alkyl, C₁₋₁₀alkoxy, substitutedC₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino,substituted amino, aminoacyl, aminocarbonyl aminocarbonylamino,aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl,substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy,C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester,(carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl,substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted(C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted(C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substitutedC₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy,C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl,C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substitutedC₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substitutedC₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol,C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

Or any R⁴ forms a cyclic structure formed with any R³, the cyclicstructure is selected from the group consisting of C₂₋₁₀heterocyclyl andC₁₋₁₀heteroaryloptionally substituted with H, halogen, hydroxyl, N₃,NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl substitutedC₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino,substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl,aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy,aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy,substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio,carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano,C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy,substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted(C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substitutedC₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy,C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl,C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substitutedC₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substitutedC₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol,C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

n is an integer selected from 0 to 4; m is an integer selected from 0 to5; each p is an integer independently selected from 0 to 2; q is aninteger selected from 1 to 10.

In some embodiments, q can be 1. In some embodiments, q can be 2. Insome embodiments, q can be 3. In some embodiments, q can be 4. In someembodiments, q can be 5. In some embodiments, q can be 6. In someembodiments, q can be 7. In some embodiments, q can be 8. In someembodiments, q can be 9. In some embodiments, q can be 10. In specificembodiments, q is 3 or 4.

In some embodiments, the Rapafucin compounds in the present disclosurecan have a structure according to Formula (VI) or an optically purestereoisomer or pharmaceutically acceptable salt thereof.

Each L¹, L², or L³ can be independently selected from the linkerstructures in Table 1. Each AA₁, AA₂, AA₃, or AA₄ can be independentlyselected from the amino acid monomers shown in Table 3 below. X can beCH₂, NH, O, or S; Y can be O, NH, or N-alkyl; E can be CH or N; n is aninteger selected from 0 to 4. Amino acids can be either N—C linked orC—N linked.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl,C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, andCO₂C₁₋₂₀alkyl. R² is selected from the group consisting of C₆₋₁₅aryl andC₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃,NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy,substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy,amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl,aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy,aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy,substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio,carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano,C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy,substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted(C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl,C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio,substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substitutedheterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl.

V is

Z is a bond,

wherein R³ and R⁴ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁵,CR⁵, N, and NR⁵, wherein R⁵ is hydrogen or alkyl.

Synthetic route to Rapafucins. There are several methods for thesynthesis of rapafucins including both solid and solution phasesynthesis. These methods can result in modifications to the linker(s)and/or the effector domain which include alkylations, amide bondformations, double bond metathesis, oxadiazole formation, triazoleformations, dithiol formations, sulfone formations, Diels-Aldercycloadditions, and others.

We applied solid-phase peptide synthesis to assemble the polypeptideeffector domains. The pre-assembled FKBD capped with a carboxylic acidat one end and an olefin at the other was subsequently coupled to thepolypeptide that remained tethered on beads. To facilitate purificationof the newly formed macrocycles, we adopted a coupled macrocyclizationand cyclative release strategy whereby the macrocyclization isaccompanied by the concurrent release of the macrocyclic products fromthe solid beads. One skilled in the art can contemplate differentmacrocyclization methods for the synthesis of Rapafucin molecules in thepresent disclosure. In some embodiments, a ring-closingmetathesis/cyclative release (RCM) is used. In some embodiments,macrolactamization can be used for efficient parallel synthesis ofdifferent Rapafucins. A cis-C₆ linker can be used for construction ofRapafucin libraries. A combination of medium temperature and catalystloading (140° C., 30 mol % Hoveyda-Grubbs II catalyst) for the ensuinglarge-scale synthesis of Rapafucin libraries.

Other ring-closing methods can be used to synthesize the Rapafucinmolecules disclosed herein. Exemplary methods can include, but notlimited to aminolysis, chemoenzymatic method, click chemistry,macrocylization through ring contraction using auxiliary groups,macrocylization mediated through sulfur containing groups,macrocylization via cycloaddition, macrocylization via Wittiga or Wittiglike reactions, macrocylization from multicomponent reactions,metal-assisted macrocylization, macrocylization through C—N bondformation, macrocylization through C—O bond formation, alkylation withor without metal assistance, intramolecular cyclopropanation, oxidativecoupling of arenes, side chain cyclization, and oxidative coupling ofarenes. Each of these macrocyclization method can be conducted in solidphase or solution phase. The macrocyclization reactions through ringcontraction using auxiliary groups can include, but not limited to usinghydroxyl benzaldehyde, using hydroxyl nitro phenol, and using nitrovinyl phenol. The macrocylization reactions mediated through sulfurcontaining groups can include, but not limited to thiazolidine formationO to N acyl transfer, transesterification S to N acyl transfer, ringchain tautomerization S to N acyl transfer, Staudinger ligation ringcontraction, bis-thiol-ene macrocyclization, thiol-ene macrocyclization,thiolalkylation, and disulfide formation. The macrocyclization reactionsvia cycloaddtion can include, but not limited to phosphorene-azideligation and oxadiazole graft. Metal assisted macrocyclization caninclude, but not limited to C—C bond formation, Suzuki coupling,Sonogashira coupling, Tasuji-Trost reaction, Glaser-Hay coupling, andNickel catalyzed macrocylication. Macrocyclization reactions via C—Nbond formation can include, but not limited to Ullmann coupling andBuchwald-Hartwig animation. Macrocyclization reactions via C—O bondformation can include, but not limited to Chan-Lam-Evans coupling, C—Hactivation, and Ullmann coupling. Macrocyclization reactions viaalkylation can include enolate chemistry, Williamson etherification,Mitsunobu reaction, aromatic nucleophilic substitution (SNAr), andFriedel-Crafts type alkylation.

In some embodiments, Rapafucin molecules can be cyclized using themethods described in Marsault, E., & Peterson, M. L. (Eds.). (2017).Practical Medicinal Chemistry with Macrocycles: Design, Synthesis, andCase Studies, which is hereby incorporate d by reference in itsentirety. Some non-limiting examples of the macrocyclization methods areshown in Table 2 below, each n can be independently an integer selectedfrom 0 to 10.

TABLE 2 Additional macrocyclization methods that can be used forRapafucin synthesis. Macro- cyclization reactions Reaction schemeCyclization by intra- molecular aminolysis

Macro- cyclization via Chemo- enzymatic methods

Cyclization by intra- molecular aminolysis- II

Macro- cyclization through ring contraction using auxiliary groups-using hydroxyl benzal- dehyde

Macro- cyclization through ring contraction using auxiliary groups-using hydroxyl nitro phenol

Macro- cyclization through ring contraction using auxiliary groups-using nitro vinyl phenol

Macro- cyclization mediated through sulfur containing groups- viathiazo- lidine formation O to N acyl transfer

Macro- cyclization mediated through sulfur containing groups-viatransester- ification S to N acyl transfer

Macro- cyclization mediated through sulfur containing groups- via ringchain tautomeri- zation S to N acyl transfer

Macro- cyclization mediated through sulfur containing groups- Staudingerligation ring contraction

Macro- cyclization mediated through sulfur containing groups-bisthiolene macro- cyclization

Macro- cyclization mediated through sulfur containing groups- thiol-enemacro- cyclization

Macro- cyclization mediated through sulfur containing groups- thio-alkylation

Macro- cyclization mediated through sulfur containing groups- disulfideformation

Macro- cyclization via cyclo- addition- phospho- rene-azide ligation

Macro- cyclization via azide- alkyne cyclo- addition- 1,3-dipolarHuisgen cyclo- addition

Macro- cyclization via cyclo- addition- oxadiazole graft using (N-iso-cyanimino) triphenyl- phospho- rane

Macro- cyclization via Wittig or Horner- Wadsworth- Emmons or Masamune-Roush reactions or Still- Gennari olefination

Macro- cyclization from multi- component reactions

Metal assisted macro- cyclization- C—C bond formation (Metals includePd, Ni, Cu, Ru, or Au)

Metal assisted macro- cylization C═C bond formation (Metals include Pd,Ni, Cu, Ru, or Au)

Metal assisted macro- cyclization- Suzuki coupling

Metal assisted macro- cyclization- Sono- gashira coupling

Metal assisted macro- cyclization- Tsuji- Trost reaction

Metal assisted macro- cyclization- Glaser- Hay coupling

Metal assisted macro- cyclization- Nickel catalyzed macro- cyclization

Macro- cyclization via C—N bond formation- Ullmann coupling

Macro- cyclization via C—N bond formation- Buchwald- Hartwig amination

Macro- cyclization via C—N bond formation- Chan- Lam- Evans coupling

Macro- cyclization via C—N bond formation- C—H activation

Macro- cyclization via C—N bond formation- Ullmann coupling

Macro- cyclization via alkylation- enolate chemistry

Macro- cyclization via alkylation- William- son etherifi- cation

Macro- cyclization via alkylation- Mitsunobu reaction

Macro- cyclization via alkylation- aromatic nucleo- philic substi-tution (SNAr)

Macro- cyclization via alkylation- Friedel- Crafts type alkyl- ations

Macro- cyclization through intra- molecular cyclopro- panation

Macro- cyclization through oxidative coupling of Arenes

Macro- cyclization- side chain cyclization

Macro- cyclization- oxidative coupling of arenes

In some embodiments, the Rapafucin compounds in the present disclosurecan have a structure according to Formula (VII) or an optically purestereoisomer or pharmaceutically acceptable salt thereof.

Each T₁ or T₂ can be independently selected from the terminal structuresas outlined in Table 2 above before macrocyclization. Each L₁, L₂, or L₃can be independently selected from the linker structures in Table 1.Each AA can be independently selected from the amino acid monomers shownin Table 3 below. X can be CH₂, NH, O, or S; Y can be O, NH, or N-alkyl;E can be CH or N; n is an integer selected from 0 to 4. Amino acids canbe either N—C linked or C—N linked.

In some embodiments, m can be 1. In some embodiments, m can be 2. Insome embodiments, m can be 3. In some embodiments, m can be 4. In someembodiments, m can be 5. In some embodiments, m can be 6. In someembodiments, m can be 7. In some embodiments, m can be 8. In someembodiments, m can be 9. In some embodiments, m can be 10. In a specificembodiment, m is 3 or 4.

V is

Z is a bond,

wherein R³ and R⁴ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁵,CR⁵, N, and NR⁵, wherein R⁵ is hydrogen or alkyl.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl,C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, andCO₂C₁₋₂₀alkyl. R² is selected from the group consisting of C₆₋₁₅aryl andC₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃,NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy,substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy,amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl,aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy,aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy,substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio,carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano,C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy,substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted(C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl,C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio,substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substitutedheterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl.

Table 3 below shows the FKBD moieties with linkers before incorporatedinto the Rapafucin macrocylic structure.

TABLE 3 The FKBD/linker moieties used in the present disclosure. FKBDidentifier Chemical Structure aFKBD

eFKBD

Raa1

Raa2

Raa3

Raa4

Raa5

Raa6

Raa7

Raa8

Raa9

Raa10

Raa11

Raa12

Raa13

Raa14

Raa15

Raa16

Raa17

Raa18

Raa19

Raa20

Raa21

Raa22

Raa25

Raa26

Raa27

Raa28

Raa29

Raa30

Rae1

Rae2

Rae3

Rae4

Rae5

Rae9

Rae10

Rae11

Rae12

Rae13

Rae14

Rae15

Rae16

Rae17

Rae18

Rae19

Rae20

Rae21

Rae22

Rae23

Rae24

Rae25

Rae26

Rae27

Rae28

Rae29

Rae30

Rae31*

Rae32

Rae33

Rae34

Rae35

Rae36

Rae37

Rae38

*This FKBD is reduced and cyclized via lactamization.

Table 4 below shows the amino acid monomers used for the the Rapafucinmacrocylic compounds synthesis in the present disclosure.

TABLE 4 The monomers used in the present disclosure. Entry Monomer No.identifier Chemical Structure  1 G

 2 Sar

 3 dA

 4 A

 5 bAla

 6 Dpr

 7 ra199

 8 mA

 9 Alb

 10 Abu

 11 C

 12 dC

 13 SeC

 14 DSec

 15 dS

 16 S

 17 ra165

 18 Aze

 19 ra126

 20 ra524

 21 dP

 22 P

 23 ra132

 24 SbPro

 25 RbPro

 26 ra603

 27 Dab

 28 ra484

 29 ra203

 30 ra201

 31 ra202

 32 isoV

 33 ra130

 34 Nva

 35 ra131

 36 dV

 37 V

 38 bVal

 39 Hcy

 40 mC

 41 dT

 42 T

 43 mS

 44 Hse

 45 Bux

 46 Om

 47 dN

 48 N

 49 RbAsn

 50 SbAsn

 51 RbAsp & dD

 52 D

 53 ra344

 54 mV

 55 ra345

 56 ra379

 57 ra359

 58 Nle

 59 Dl

 60 L

 61 dI

 62 I

 63 Tle

 64 Rblle

 65 Sblle

 66 SbLeu

 67 RbLeu

 68 ra74

 69 RbMet

 70 SbMet

 71 M

 72 dM

 73 Pen

 74 ra371

 75 mT

 76 ra582

 77 ra380

 78 ra473

 79 ra341

 80 ra538

 81 ra555

 82 ra550

 83 Spg

 84 ra144

 85 ra189

 86 ra330

 87 ra541

 88 ra528

 89 ra168

 90 ra532

 91 Roh4P

 92 ra508

 93 ra557

 94 ra576

 95 Glp

 96 ra505

 97 ra518

 98 ra584

 99 ra372

100 ra83

101 ra162

102 ra169

103 ra127

104 ra76

105 ra600

106 ra128

107 ra564

108 ra510

109 ra464

110 ra466

111 ra543

112 ra170

113 m4oh3P

114 dK

115 K

116 SbLys

117 RbLys

118 mN

119 dQ

120 Q

121 RbGln

122 SbGln

123 mD

124 dE

125 E

126 ra206

127 RbGlu

128 mI

129 ra352

130 ra147

131 ra207

132 mL

133 ra530

134 Elscy

135 mM

136 ra61

137 Cya

138 ra401

139 mK

140 oh5K

141 mQ

142 mE

143 Aad

144 ra458

145 ra459

146 ra583

147 ra310

148 ra563

149 Tza

150 ra301

151 ra507

152 ra509

153 ra602

154 ra601

155 Phg

156 ra84

157 ra337

158 ra338

159 ra363

160 ra364

161 Thl

162 ra368

163 ra67

164 ra68

165 dH

166 H

167 SbHis

168 RbHis

169 ra405

170 ra90

171 ra406

172 ra89

173 ra91

174 ra176

175 ra462

176 ra461

177 ra565

178 ra122

179 dF

180 F

181 ra527

182 Cha

183 SbPhe

184 RbPhe

185 ra516

186 ra325

187 ra450

188 ra522

189 mH

190 Hhs

191 ra490

192 ra609

193 ra173

194 ra102

195 ra542

196 Olc

197 ra540

198 dR

199 R

200 RbArg

201 SbArg

202 Apm

203 ra355

204 ra300

205 ra581

206 ra142

207 ra183

208 ra562

209 Sta

210 Cit

211 mR

212 Har

213 ra664

214 Dpm

215 m3K

216 Ra590

217 ra307

218 ra547

219 Asu

220 ra535

221 ra348

222 Aca

223 Gla

224 ra80

225 ra545

226 Tic

227 ra351

228 ra350

229 ra69

230 ra101

231 ra204

232 ra521

233 ra523

234 ra172

235 ra195

236 mF

237 ra558

238 ra120

239 ra659

240 ra134

241 ra59

242 ra549

243 ra104

244 ra123

245 ra87

246 ra336

247 ra116

248 ra665

249 ra117

250 ra115

251 ral118

252 ra339

253 ra119

254 ra666

255 ra121

256 ra551

257 ra539

258 ra381

259 dY

260 Y

261 ra469

262 ra400

263 ra106

264 ra335

265 ra513

266 ra329

267 SbTyr

268 RbTyr

269 ra658

270 ra113

271 ra114

272 ra596

273 ra112

274 ra561

275 ra208

276 ra63

277 ra66

278 ra55

279 ra62

280 ra56

281 ra534

282 ra387

283 ra386

284 ra374

285 ra360

286 ra64

287 ra65

288 ra382

289 ra537

290 ra88

291 ra209

292 ra497

293 ra185

294 mY

295 ra133

296 ra667

297 ra124

298 Uraa1

299 ra594

300 Dsu

301 ra456

302 ra457

303 ra589

304 ra559

305 ra536

306 ra548

307 ra573

308 ra86

309 ra574

310 ra533

311 ra75

312 ra105

313 ra136

314 ra454

315 ra321

316 ra588

317 ra560

318 ra517

319 ra648

320 ra317

321 ra302

322 ra660

323 ra108

324 ra378

325 ra109

326 ra597

327 ra111

328 ra579

329 App

330 Cap

331 dW

332 W

333 SbTrp

334 RbTrp

335 ra347

336 ra575

337 ra404

338 ra407

339 ra129

340 ra608

341 ra642

342 ra463

343 ra467

344 ra529

345 ra468

346 ra140

347 ra141

348 no22Y

349 ra591

350 ra638

351 ra650

352 ra592

353 ra578

354 ra604

355 ra373

356 ra171

357 ra110

358 ra107

359 ra93

360 ra370

361 ra92

362 ra79

363 ra639

364 ra649

365 ra546

366 ra554

367 mW

368 ra324

369 ra327

370 ra605

371 Ra385

372 ra354

373 ra58

374 ra314

375 ra486

376 ra567

377 napA

378 ra566

379 ra148

380 ra167 & ra78

381 ra71

382 ra334 & ra487

383 ra333

384 ra452

385 ra306

386 ra637

387 ra587

388 ra586

389 ra643

390 ra453

391 ra308

392 ra305

393 ra661

394 ra647

395 ra326

396 ra323

397 ra342

398 ra496

399 ra332

400 ra593

401 ra81

402 ra663

403 ra640

404 ra646

405 ra636

406 ra652

407 ra515

408 ra520

409 ra94

410 ra137

411 ra495 & ra531

412 ra641

413 ra651

414 ra612

415 ra500

416 ra644

417 ra399

418 ra98

419 ra645

420 Pyl

421 DPyl

422 ra662

423 ra653

424 ra491

425 ra577

426 ra70

427 ra95

428 ra97

429 ra136

430 ra96

431 ra514

432 ra654

433 ra657

434 ra511

435 ra366

436 pnaC

437 ra615

438 pnaT

439 ra624

440 ra526

441 ra525

442 ra471

443 ra613

444 ra599

445 ra553

446 ra626

447 ra633

448 ra628

449 ra60

450 ra73

451 ra175

452 ra606

453 ra398

454 ra494

455 ra501

456 ra503

457 ra611

458 ra353

459 ra616

460 ra629

461 ra504

462 pnaA

463 ra318

464 ra614

465 ra630

466 ra512

467 ra319

468 Pqa

469 ra619

470 ra627

471 ra623

472 ra358

473 ra346

474 ra492

475 ra493

476 ra617

477 ra622

478 ra502

479 ra655

480 ra618

481 ra625

482 ra621

483 ra631

484 pnaG

485 ra607

486 ra656

487 ra620

488 ra668

489 ra635

490 ra472

491 ra569

492 ra632

493 ra634

494 ra570

495 ra595

496 ra311

497 ra304

498 ra303

499 ra571

500 ra309

501 ra402

502 ra322

503 ra349

504 ra408

505 ra572

506 ra580

507 Ala

508 mAla

509 dAla

510 ChA

511 Pro

512 Val

513 mVal

514 Leu

515 dLeu

516 mLeu

517 mdLeu

518 mLeu

519 HoSerMe

520 Phe

521 Nal

522 Nva

523 PhF

524 PhG

525 dPhe

526 mG

527 mNle

528 mPhe

529 mSerBu

530 mdPhe

531 mlle

The monomers RbAsp, dD, D, and SbAsp have more than one hydroxyl groups.In some embodiments, the hydroxyl group that serves as a linkage pointto the adjacent residues in each of these monomers is illustrated inScheme 2 above. In some embodiments, the other hydroxyl group in thesemonomers can be used as a linkage point to the adjacent residues.

In some embodiments, disclosed herein is a compound of Formula VIII or apharmaceutically acceptable salt or solvate thereof.

In some embodiments, R can be

R¹, R², R³, R⁴, and R⁵ can be each independently selected from hydrogen,hydroxyl, alkoxy, cyano, alkylthio, amino, and alkylamino, and

wherein

can be a resin; wherein one, two, three, or four of A¹, A², A³, A⁴, andA⁵ can be N or P with the remaining being CH; wherein one, two, three,or four of B¹, B², B³ and B⁴ can be O, N, or S with the remaining beingCH or CH₂ as appropriate; wherein

can be a single or double bond.

In some embodiments, X₁ can be O or NR⁶; Y can be —C(O)— or

X₂ can be (CH₂)_(m), O, OC(O), NR⁶, NR⁶C(O); Z can be

W can be O, CH, CH₂, CR⁹, or C R¹⁰R¹¹; can be L₁ and L₂ can be eachindependently a direct bond, substituted or unsubstituted−(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-,substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted orunsubstituted —(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted orunsubstituted —(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted orunsubstituted —(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, —O—, —NH—, —S—, —S(O)—,—SO₂—, —Si—, and —B—, wherein each alkyl, alkenyl, and alkynyl group maybe optionally substituted with alkyl, alkoxy, amino, hydroxyl,sulfhydryl, halogen, carboxyl, oxo, cyano, nitro, or trifluoromethyl.

L₃ can be a direct bond, substituted or unsubstituted —(C₁-C₆)alkyl-,substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alky-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted—(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted orunsubstituted —(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted orunsubstituted —(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted—(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted orunsubstituted —(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, wherein each alkyl,alkenyl and alkynyl group may be optionally substituted with alkyl,alkoxy, amino, hydroxyl, sulfhydryl, halogen, carboxyl, oxo, cyano,nitro, or trifluoromethyl.

Each m can be independently an integer selected from 0, 1, 2, 3, 4, 5,and 6; each n is independently an integer selected from 0, 1, 2, 3, 4,5, and 6; R⁶ is hydrogen or alkyl; R⁷ and R⁸ are each independentlyselected from hydrogen, hydroxy, alkyl, alkoxy, cyano, alkylthio, amino,and alkylamino, and OPG, wherein OPG is a protecting group; R⁹, R¹⁰, andR¹¹ are each independently selected from hydrogen, hydroxy, alkyl,alkoxy, cyano, alkylthio, amino, and alkylamino, and OPG, wherein OPG isa protecting group.

The Effector Domain can have Formula (A):

R¹², R¹⁴, R¹⁶, and R¹⁸ can be each independently hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted orunsubstituted alkoxy, substituted or unsubstituted alkylamino,substituted or unsubstituted aryl, substituted or unsubstitutedalkylaryl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl,substituted or unsubstituted heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃,(CH₂)_(n)C₂F₅. R¹³, R¹⁵, and R¹⁷ are each independently the sidechainsof naturally occurring amino acids and their modified forms includingbut are not limited to D-amino acid configuration, or hydrogen, halogen,amino, cyano, nitro, trifluoromethyl, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted perfluoroalkyl, substituted orunsubstituted alkoxy, substituted or unsubstituted alkylamino,substituted or unsubstituted alkylthio, substituted or unsubstitutedaryl, substituted or unsubstituted alkylaryl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted heteroalkylaryl, substituted or unsubstituted(CH₂)_(n)-aryl, substituted or unsubstituted (CH₂)_(n)-heteroaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹,(CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹,(CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹,(CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)SR¹⁹,(CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)_(j)R¹⁹, or—(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹.

R¹² and R¹³, R¹⁴ and R¹⁵, R¹⁶ and R¹⁷ can be convalently connected toform a substituted or unsubstituted 5-, 6-, or 7-membered heterocycle.Each k can be independently an integer selected from 0, 1, 2, 3, 4, 5,6, 7, 8, 9, and 10. Each j can be independently an integer selected from0, 1, and 2. R¹⁹, R²⁰, R²¹, and R²² can be each independently hydrogen,halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl,cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl,alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, orheteroalkylaryl.

Or R¹⁹ and R²² are as described above, and R²⁰ and R²¹, together withthe N atom to which they are attached, form a substituted orunsubstituted 5-, 6-, or 7-membered heterocycloalkyl or a substituted orunsubstituted 5-membered heteroaryl, wherein each of the above groupslisted for R¹³, R¹⁵, and R¹⁷ may be optionally independently substitutedwith 1 to 3 groups selected from halogen, amino, cyano, nitro,trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl,alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl,heterocycloalkyl, heteroaryl, heteroalkylaryl, (CH₂)_(n)CN,(CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹, (CH₂)_(n)C(O)R¹⁹,(CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹, (CH₂)_(n)NR²⁰R²¹,(CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹, (CH₂)_(n)NR²²C(O)OR¹⁹,(CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)SR¹⁹, (CH₂)_(n)S(O)_(j)NR²⁰R²¹,(CH₂)_(n)NR²²S(O)_(j)R¹⁹, or —(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹.

Or the Effector Domain can have Formula (B):

Each k can be independently an integer selected from 0, 1, 2, 3, 4, 5,6, 7, 8, 9, and 10; R²³ can be a hydrogen or alkyl; X₃ can besubstituted or unsubstituted —(C₁-C₃₀)alkyl-, alkenyl-, alkynyl- witheach carbon individually assuming one of the following redox states:CH₂, CH—OH, C(O);

Or the Effector Domain can have Formula (C):

X₄ can be substituted or unsubstituted —(C₁-C₃o)alkyl-, alkenyl-,alkynyl- with each carbon individually assuming one of the followingredox states: CH₂, CH—OH, C(O).

Or the Effector Domain has Formula (D):

R²⁴ and R²⁵ are each a hydrogen or alkyl; X₅ can be substituted orunsubstituted —(C₁-C₃₀)alkyl-, alkenyl-, alkynyl- with each carbonindividually assuming one of the following redox states: CH₂, CH—OH,C(O).

Or the Effector Domain can be Formula (E):

X₆ can be substituted or unsubstituted —(C₁-C₃₀)alkyl-, alkenyl-,alkynyl- with each carbon individually assuming one of the followingredox states: CH₂, CH—OH, C(O).

In some embodiments, L₃ is not

with R²⁶ being hydrogen or alkyl.

In some embodiments, R is not

wherein R³ is hydrogen, hydroxyl, or OPG, wherein PG is a protectinggroup, or

wherein

is a resin; wherein R² is hydrogen, hydroxyl, or alkoxy; and wherein R¹,R⁴, and R⁵ are each independently hydrogen or no substituent as dictatedby chemical bonding; wherein

is a single or double bond.

In some embodiments, L₁ and L₂ not each independently direct bond,substituted or unsubstituted —(C₁-C₆)alkyl-, substituted orunsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)—, substituted or unsubstituted—(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted—(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted—(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted—(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl,and alkynyl group may be optionally substituted with alkyl, alkoxy,amino, carboxyl, cyano, nitro, or trifluoromethyl.

In some embodiments, the Effector Domain is a compound of Formula (F)

R¹², R¹⁴, R^(14′), R¹⁶, and R²⁷ are not each independently hydrogen oralkyl and R¹³, R¹⁴, R^(14′), and R¹⁶ are not each independentlyhydrogen, halogen, amino, cyano, nitro, trifluoromethyl, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted perfluoroalkyl, substituted orunsubstituted alkoxy, substituted or unsubstituted alkylamino,substituted or unsubstituted alkylthio, substituted or unsubstitutedaryl, substituted or unsubstituted alkylaryl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃,(CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹, (CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹,(CH₂)_(n)OC(O)R¹⁹, (CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹,(CH₂)_(n)NR²²C(O)R¹⁹, (CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹,(CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)_(j)R¹⁹, or—(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹; n is an integer selected from 0, 1, 2, 3,4, 5, and 6; j is an integer selected from 0, 1, and 2.

R¹⁹, R²⁰, R²¹, and R²² are each independently hydrogen, halogen, amino,cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl,perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl,heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl, or R¹⁹and R²² are as described above, and R²⁰ and R²¹, together with the Natom to which they are attached, form a substituted or unsubstituted 5-,6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted5-membered heteroaryl.

Each of the above groups listed for R¹³, R¹⁵, and R¹⁷ may be optionallyindependently substituted with 1 to 3 groups selected from halogen,amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl,cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl,alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹,(CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹,(CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹,(CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)SR¹⁹,(CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)_(j)R¹⁹, or—(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹.

In some embodiments, L₃ in Formula (VII) is —CH₂CH₂—, R is

R¹, R⁴, R⁵ and Ware each hydrogen; R² and R³ are each methoxy; m=0; Y is

X₂ is O or NR⁶C(O); L₁ is —CH₂—C(O)— or —(CH₂)₂C(O)—; Z is

L₂ is —OCO—CH═CH—(CH₂)₂N(Me)-. In some embodiments, X₂ is O and Li is—CH₂—C(O)—. In some embodiments, X₂ is NR⁶C(O) and L₁ is —(CH₂)₂C(O)—.

In some embodiments, the effector domain can be Formula (G)

Wherein R^(12,)R¹⁴, R^(14′), and R¹⁶ are each independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl,substituted or unsubstituted alkoxy, substituted or unsubstitutedalkylamino, substituted or unsubstituted aryl, substituted orunsubstituted alkylaryl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅.

R¹³, R¹⁵, R^(15′) and R¹⁷ are each independently the sidechains ofnaturally occurring amino acids and their modified forms including butare not limited to D-amino acid configuration, or hydrogen, halogen,amino, cyano, nitro, trifluoromethyl, substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted perfluoroalkyl, substituted orunsubstituted alkoxy, substituted or unsubstituted alkylamino,substituted or unsubstituted alkylthio, substituted or unsubstitutedaryl, substituted or unsubstituted alkylaryl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted heteroalkylaryl, substituted or unsubstituted(CH₂)_(n)-aryl, substituted or unsubstituted (CH₂)_(n)-heteroaryl,(CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹,(CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹,(CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹,(CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²¹R²¹, (CH₂)_(n)SR¹⁹,(CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)_(j)R¹⁹, or—(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹. R¹² and R¹³, R¹⁴ and R¹⁵, R^(14′) andR^(15′), R¹⁶ and R¹⁷ can be covalently connected to form a substitutedor unsubstituted 5-, 6-, or 7-membered heterocycle.

In some embodiments, disclosed herein is a method of using a hybridcyclic library based on the immunophilin ligand family of naturalproducts FK506 and rapamycin, to screen for compounds for treatingcancer. In some embodiments, disclosed herein is a method of using ahybrid cyclic library based on the immunophilin ligand family of naturalproducts FK506 and rapamycin, to screen for compounds for treatingautoimmune disease.

In some embodiments, the Rapafucin compounds in the present disclosurecan have a structure according to Formula (IX) or Formula (X) or anoptically pure stereoisomer or pharmaceutically acceptable salt thereof.

The amino acid moieties with R¹, R², R³, and R⁴ can be selected fromTable 2 below illustrating the amino acid monomers used for the presentdisclosure. In some embodiments, the amino acid moieties with R¹ and R³can be selected from the group consisting of

or R₁ or R₃ together with nitrogen to form

In some embodiments, the amino acid moieties with R₂ and R⁴ can beselected from the group consisting of

The macrocyclic natural products FK506 and rapamycin are approvedimmunosuppressive drugs with important biological activities. Both havebeen shown to inhibit T-cell activation, each with distinct mechanisms.In addition, rapamycin has been shown to have strong anti-proliferativeactivity. FK506 and rapamycin share an extraordinary mode of action;they act by recruiting an abundant and ubiquitously expressed cellularprotein, the prolyl cis-trans isomerase FKBP, and the binary complexessubsequently bind to and allosterically inhibit their target proteinscalcineurin and mTOR, respectively. Structurally, FK506 and rapamycinshare a similar FKBP-binding domain but differ in their effectordomains. In FK506 and rapamycin, nature has taught us that switching theeffector domain of FK506 to that in rapamycin, it is possible to changethe targets from calcineurin to mTOR. The generation of a rapafucinlibrary of macrocyles that contain FK506 and rapamycin binding domainsshould have great potential as new leads for developing drugs to be usedfor treating diseases.

A variety of methods exist for the generation of compound libraries fordeveloping and screening potentially useful compounds in treatingdiseases. One such method is the development of encoded libraries, andparticularly libraries in which each compound includes an amplifiabletag. Such libraries include DNA-encoded libraries in which a DNA tagidentifying a library member can be amplified using molecular biologytechniques, such as the polymerase chain reaction (PCR). The use of suchmethods for producing libraries of rapafucin macrocyles that containFK506-like and rapamycin-like binding domains has yet to bedemonstrated. Thus, there remains a need for DNA-encoded rapafucinlibraries of macrocyles that contain FK506-like and rapamycin-likebinding domains.

In one aspect, provided herein is a tagged macrocyclic compound thatcomprises: an FK506 binding protein binding domain (FKBD); an effectordomain; a first linking region; and a second linking region; wherein theFKBD, the effector domain, the first linking region, and the secondlinking region together form a macrocycle; and wherein at least one ofthe FKBD, the effector domain, the first linker, and the second linkercan be operatively linked to one or more oligonucleotides (D) which canidentify the structure of at least one of the FKBD, the effector domain,the first linker, and the second linker.

In certain embodiments, provided herein is a tagged macrocyclic compoundof Formula (XI):

In some embodiments, h, i, j, and k are each independently an integerfrom 0-20, provided that at least one of h, i, j, and k is not 0; and Dis an oligonucleotide that can identify at least one of the FKBD, theEffector Domain, the Linking Region A, or the Linking Region Z, wherethe solid lines linking the FKBD, the Effector Domain, the LinkingRegion A, and/or the Linking Region Z indicate an operative linkage andthe squiggle lines indicate an operative linkage. In certainembodiments, oligonucleotide (D) can be operatively linked to at leastone of the FKBD, the Effector Domain, the Linking Region A, or theLinking Region Z.

In some embodiments, provided herein is a tagged macrocyclic compound ofFormula (XII) or a pharmaceutically acceptable salt, solvate, orstereoisomer thereof:

In some embodiments, Ring A is a 5-10 membered aryl, cycloalkyl,heteroaryl or heterocycloalkyl, optionally substituted with 1-17substituents, each of which is independently selected from the groupconsisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl,haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; J is independently at each occurrence selected from thegroup consisting of —C(O)NR⁶—.

wherein R⁶ is each hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R′ is hydrogen, alkyl,arylalkyl, or haloalkyl; D is independently at each occurrence anoligonucleotide; L^(b) and L^(c) are independently at each occurrenceselected from the group consisting of bond, —O—, —S—, —OC(O)—, —C(O)O—,—(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—,—NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆alkylene (CH₂)_(n)—,optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)nNR⁵C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)OC₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)OC(O)C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆alkylene (CH₂)n-, optionally substituted(CH₂)_(n)—S—C₁₋₆alkylene (CH₂)_(n)—, and optionally substituted(CH₂CH₂₀)_(n); wherein each alkylene is optionally substituted with 1 or2 groups independently selected from the group consisting of of halo,hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano,nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl,arylalkyl,

or and

wherein R^(N) is aryl, alkyl, or arylalkyl; X is O, S or NR⁸, wherein R⁸is hydrogen, hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ areeach independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionallysubstituted with 1-10 substituents, each of which is selected from thegroup consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano,haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen,hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is eachindependently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl,haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸,CR¹⁸, N, or and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond,—O—, —NR¹⁹—, —SO—, —SO₂—, (CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6membered heteroaryl, optionally substituted with 1-4 substituents, eachof which is independently selected from the group consisting ofhydrogen, hydroxyl, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano,alkylthio, amino, alkylamino, dialkylamino and

wherein each R¹⁹, R²⁰, and R²¹ is independently is selected from thegroup consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula(XIIa):

In some embodiments, each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f),k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b),X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently abond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃)alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i) and R⁴ isindependently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ ishydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a),R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f),R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) isindependently selected from the group consisting of hydrogen, halo,amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted alkylamino,optionally substituted dialkylamino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted arylalkyl,optionally substituted heteroarylalkyl and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnaturalamino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

wherein each t is independently an integer selected from 0, 1, 2, 3, 4,5, 6, 7, 8, 9, and 10; R²⁹ is a hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³²are each independently hydrogen or alkyl; X³ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

wherein X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

wherein R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ issubstituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-,—(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; provided that when R is

L is ethylene, X is O, W is

V is

Z is

-L⁶-L⁷-L⁸- is

then -L¹-L²-L³-L⁴-L⁵- is not

and; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c),R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g),R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring Csubstituted with at least one

or wherein at least one of the linking groups selected from Table 1 issubstituted with at least one

In another aspect, provided herein is a compound library that comprisesa plurality of distinct tagged macrocyclic compounds according to any ofthe above. In certain embodiments, provided herein is a compound librarythat comprises at least about 10² distinct tagged macrocyclic compoundsaccording to any of the above. In certain embodiments, provided hereinis a compound library that comprises from about 10² to about 10¹⁰distinct tagged macrocyclic compounds according to any of the above.

In a further aspect, provided herein is a method of making a library oftagged macrocyclic compounds as disclosed herein, the method comprisingsynthesizing a plurality of distinct tagged macrocyclic compoundsaccording to any of the above.

In a still further aspect, provided herein is a method of making atagged macrocyclic compound as disclosed herein, the method comprisingoperatively linking at least one oligonucleotide (D) to at least one ofan FKBD, an effector domain, a first linking region, and a secondlinking region, and forming a macrocyclic ring comprising the FKBD, theeffector domain, the first linking region, and the second linkingregion.

In certain embodiments, provided herein is a method of making a taggedmacrocyclic compound as disclosed herein, the method comprisingmacrocyclic compound to at least one oligonucleotide (D), themacrocyclic compound comprising an FKBD, an effector domain, a firstlinking region, and a second linking region, wherein the FKBD, theeffector domain, the first linking region, and the second linking regiontogether form a macrocycle; and wherein the at least one oligonucleotide(D) can identify the structure of at least one of the FKBD, the effectordomain, the first linking region, and the second linking region.

In yet a further aspect, the method of making a tagged macrocycliccompound comprises: operatively linking a compound of Formula (XIII):

to a compound of Formula (XIV):

Q′-L^(c)-D   Formula (XIV)

In some embodiments,

and

are independently at each occurrence: a bond, —O—, —NR¹⁹—, —SO—, —SO₂—,—(CH₂)_(n)—,

or a linking group selected from Table 1 wherein Ring C is a 5-6membered heteroaryl, optionally substituted with 1-4 substituents, eachof which is independently selected from the group consisting ofhydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano,alkylthio, amino, alkylamino, dialkylamino ; wherein R¹⁹ is selectedfrom the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl; Q and Q′ are eachindependently selected from the group consisting of N₃, —C≡CH, NR⁶R⁷,—COOH, —ONH₂, —SH, —NH₂,

—(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl,arylalkyl, or haloalkyl; L^(b) and L^(c) are independently at eachoccurrence selected from the group consisting of a bond, —O—, —S—,—OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—,—(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted(CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)—S—C₁₋₆ alkylene-(CH₂)_(n)—, and optionally substituted(CH₂CH₂₀)_(n); wherein each alkylene is optionally substituted with 1 or2 groups independently selected from the group consisting of of halo,hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano,nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl;

D is an oligonucleotide; h, i, j, and k are each independently aninteger from 0-20, provided that at least one of h, i, j, and k is not0; n is an integer from 1-5; m is an integer from 1-5.

In another aspect, provided herein is a method of making a taggedmacrocyclic compound, the method comprising operatively linking acompound of Formula (XII):

with a compound of Formula (XIV):

Q′-L^(c)-D   Formula (XIV)

Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl orheterocycloalkyl, optionally substituted with 1-17 substituents, each ofwhich is independently selected from the group consisting of hydrogen,hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio,oxo, amino, alkylamino, dialkylamino,

wherein

is a resin;

L^(b) and L^(c) are independently selected from the group consisting ofa bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—,—(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—,optionally substituted (CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionallysubstituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)—S—C₁₋₆alkylene-(CH₂)_(n)—, and optionally substituted(CH₂CH₂₀)_(n); wherein each alkylene is optionally substituted with 1 or2 groups independently selected from the group consisting of of halo,hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano,nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl;

Q and Q′ are independently selected from the group consisting of —N₃,—C≡CH, NR⁶R⁷, —COOH, —ONH₂, —SH, —NH₂,

—(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl,arylalkyl, or haloalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen,hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ areeach independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionallysubstituted with 1-10 substituents, each of which is selected from thegroup consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano,haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen,hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is eachindependently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl,haloalkoxy, aryl, arylalkyl, or heteroaryl;

Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸,CHR¹⁸, CR¹⁸, N, and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond,—O—, —NR¹⁹—, —SO—, —SO₂—, —(CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6membered heteroaryl, optionally substituted with 1-4 substituents, eachof which is independently selected from the group consisting ofhydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano,alkylthio, amino, alkylamino, dialkylamino and

wherein R¹⁹ is selected from the group consisting of hydrogen, hydroxy,OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula(XIIa):

each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i)is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f),X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—,—S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄)alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴is independently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ ishydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a),R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f),R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) isindependently selected from the group consisting of hydrogen, halo,amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted alkylamino,optionally substituted dialkylamino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted arylalkyl,optionally substituted heteroarylalkyl, and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnaturalamino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7,8, 9, and 10; R²⁹ is hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³²are each independently hydrogen or alkyl; X³ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; and provided that when RingA is

L^(a) is ethylene, X is O, W is

V¹ is

V² is

Z is

-L⁶-L⁷-L⁸- is

and -L¹-L²-L³-L⁴-L⁵- is not

D is an oligonucleotide; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c),R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g),R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring Csubstituted with at least one

or wherein at least one of the linking groups selected from Table 1 issubstituted with at least one

In yet another aspect, provided herein is a method for identifying oneor more compounds that bind to a biological target the methodcomprising: (a) incubating the biological target with at least a portionof the plurality of distinct tagged macrocyclic compounds of thecompound library of claim 2 to make at least one bound compound and atleast one unbound compound of the plurality of distinct taggedmacrocyclic compounds; (b) removing the at least one unbound compound;and (c) sequencing each of the oligonucleotides (D) of the at least onebound compound.

In certain embodiments, the DNA-encoded library can be a singlepharmacophore library, wherein only one chemical moiety can be attachedto a single strand of DNA, as described in, e.g., Neri & Lerner, Annu.Rev. Biochem. (2018) 87:5.1-5.24, which is hereby incorporated byreference in its entirety. In certain embodiments, the DNA-encodedlibrary can be a dual pharmacophore library, wherein two independentmolecules can be attached to the double strands of DNA, as described in,e.g., Id; Mannocci et al., Chem. Commun. (2011) 47:12747-53, which ishereby incorporated by reference in its entirety.

In a further aspect, provided herein is a method of making a library oftagged macrocyclic compounds, the method comprising synthesizing aplurality of distinct tagged macrocyclic compounds. In certainembodiments, each tagged macrocyclic compound of the plurality ofdistinct tagged macrocyclic compounds comprising a macrocyclic compoundoperatively linked to at least one oligonucleotide (D). In certainembodiments, each compound of the plurality of distinct taggedmacrocyclic compounds of the compound library comprises a macrocycliccompound operatively linked to at least one oligonucleotide (D). Incertain embodiments, the macrocyclic compound comprising an FKBD, aneffector domain, a first linking region, and a second linking region. Incertain embodiments, the FKBD, the effector domain, the first linkingregion, and the second linking region together form a macrocycle. Incertain embodiments, each of the at least one oligonucleotide (D) canidentify at least one of the FKBD, the effector domain, the firstlinking region, and the second linking region of each of the pluralityof distinct tagged macrocyclic compounds. In certain embodiments, eachcompound of the plurality of distinct tagged macrocyclic compounds ofthe compound library comprises a compound of Formula (A) (asabove-defined). In certain embodiments, each compound of the pluralityof distinct tagged macrocyclic compounds of the compound librarycomprises a compound of Formula (I) (as above-defined herein). Incertain embodiments, each compound of the plurality of distinct taggedmacrocyclic compounds of the compound library can be a reaction productof operatively linking a compound of Formula (B) (as above-definedherein) with a compound of Formula (C) (as above-defined herein). Incertain embodiments, each compound of the plurality of distinct taggedmacrocyclic compounds of the compound library can be a reaction productof operatively linking a compound of Formula (B′) (as above-definedherein) with a compound of Formula (C) (as above-defined herein).

In certain embodiments, the method of synthesizing a library ofcompounds can be selected from the group consisting of thesplit-and-pool method, DNA-templated library synthesis (DTS), encodedself-assembling chemical (ESAC) library synthesis, DNA-recorded librarysynthesis, DNA-directed library synthesis, DNA-routing, and 3-Dproximity-based library synthesis (YoctoReactor). As a person ofordinary skill in the art would be aware, various techniques forsynthesizing the library of tagged macrocyclic compounds are describedin, e.g., Neri & Lerner, Annu. Rev. Biochem. (2018) 87:5.1-5.24; Romanet al., SLAS Discov. (2018) 23(5):387-396; Lim, C&EN, (2017) 95(29):10-10; Halford, C&EN, (2017) 95(25): 28-33; Estevez, Tetrahedron:Asymmetry. (2017) 28:837-842; Neri, Chembiochem. (2017) 4;18(9):827-828;Yuen & Franzini, Chembiochem. (2017) 4; 18(9):829-836; Skopic et al.,Chem Sci. (2017) 1;8(5):3356-3361; Shi et al.; Bioorg Med Chem Lett.(2017) 1; 27(3):361-69; Zimmermann & Neri, Drug Discov Today. (2016)21(11):1828-1834; Satz et al., Bioconjug Chem. (2015) 19; 26(8):1623-32;Ding et al., ACS Comb Sci. (2016) 10;18(10):625-629; Arico-Muendel,MedChemComm, (2016) 7(10): 1898-1909; Skopic, MedChemComm, (2016) 7(10):1957-1965; Satz, CS Comb. Sci. (2016) 18 (7):415-424; Tian et al.,MedChemComm, (2016) 7(7): 1316-1322; Salamon et al., ACS Chem Biol.(2016) 19;11(2):296-307; Satz et al., Bioconjug Chem. (2015) 19;26(8):1623-32; Connors et al., Curr Opin Chem Biol. (2015) 26:42-7;Blakskjaer et al., Curr Opin Chem Biol. (2015) 26:62-71; Scheuermann &Neri, Curr Opin Chem Biol. (2015) 26:99-103; Franzini et al., Angew ChemInt Ed Engl. (2015) 23; 54(13):3927-31; Franzini et al., Bioconjug Chem.(2014) 20;25(8):1453-61; Franzini, Neri & Scheuermann, Acc Chem Res.(2014) 15;47(4):1247-55; Mannocci et al., Chem. Commun. (2011)47:12747-53; Kleiner et al., Chem Soc Rev. (2011) 40(12): 5707-17;Clark, Curr Opin Chem Biol. (2010) 14(3):396-403; Mannocci et al., ProcNatl Acad Sci USA. (2008) 18;105(46):17670-75; Buller et al., Bioorg MedChem Lett. (2008) 18(22):5926-31; Scheuermann et al., Bioconjugate Chem.(2008) 19:778-85; Zimmerman et al., ChemBioChem (2017) 18(9):853-57, andCuozzo et al., ChemBioChem (2017), 18(9):864-71, each of which is herebyincorporated by reference in its entirety.

In some embodiments, the method of synthesizing a library of taggedmacrocyclic compounds comprises DNA-recorded library synthesis, in whichencoding and library synthesis take place separately, as described in,e.g., Shi et al., Bioorg Med Chem Lett. (2017) 1;27(3):361-369; Kleineret al., Chem Soc Rev. (2011) 40(12): 5707-17. In certain embodiments,the DNA-recorded library synthesis c comprises split-and-pool methods,which are described in, e.g., Krall, Scheuermann & Neri, Angew Chem.Int. Ed Engl. (2013) 28;52(5):1384-402; Mannocci et al., Chem. Commun.(2011) 47:12747-53; and U.S. Pat. No. 7,989,395 to Morgan et al., eachof which is hereby incorporated by reference in its entirety. In certainembodiments, the split-and-pool method comprises successive chemicalligation of oligonucleotide tags to an initial oligonucleotide (orheadpiece), which can be covalently linked to a chemically generatedentity by successive split-and-pool steps. In certain embodiments,during each split step, a chemical synthesis step can be performed alongwith an oligonucleotide ligation step.

In some embodiments, the library can be synthesized by a sequence ofsplit-and-pool cycles, wherein an initial oligonucleotide (or headpiece)can be reacted with a first set of building blocks (e.g., a plurality ofFKBD building blocks). For each building block of the first set ofbuilding blocks (e.g., each FKBD building block), an oligonucleotide (D)can be appended to the initial oligonucleotide (or headpiece) and theresulting product can be pooled (or mixed), and subsequently split intoseparate reactions. Subsequently, in certain embodiments, a second setof building blocks (e.g., a plurality of effector domain buildingblocks) can be added, and an oligonucleotide (D) can be appended to eachbuilding block of the second set of building blocks. In certainembodiments, each oligonucleotide (D) identifies a distinct buildingblock.

In some embodiments, the method of synthesizing a library of taggedmacrocyclic compounds comprises DNA-directed library synthesis, in whichDNA both encodes and templates library synthesis as described in, e.g.,Kleiner et al., Bioconjugate Chem. (2010) 21, 1836-41; and Shi et al.,Bioorg Med Chem Lett. (2017) 1;27(3):361-369, each of which is herebyincorporated by reference in its entirety. In certain embodiments, theDNA-directed library synthesis comprises the DNA-templated synthesis(DTS) method as described in, e.g., Mannocci et al., Chem. Commun.(2011) 47:12747-53, Franzini, Neri & Scheuermann, Acc Chem Res. (2014)15;47(4):1247-55; and Mannocci et al., Chem. Commun. (2011) 47:12747-53,each of which are hereby incorporated by reference in its entirety. Incertain embodiments, the DTS method comprises DNA oligonucleotides thatnot only encode but also direct the construction of the library. SeeBuller et al., Bioconjugate Chem. (2010) 21, 1571-80, which is herebyincorporated by reference in its entirety. In certain embodimentsdifferent building blocks can be incorporated into molecules usingDNA-linked reagents that can be forced into proximity by base pairingbetween their DNA tags. See Gartner et al., Science (2004) 305:1601-05,which is hereby incorporated by reference in its entirety. In certainembodiments, a library of long oligonucleotides can be synthesized firstas a template for the DNA-encoded library. In certain embodiments, theoligonucleotides can be subjected to sequence-specific chemicalreactions through immobilization on resin tagged with complementary DNAsequences. See Wrenn & Harbury, Annu. Rev. Biochem. (2007) 76:331-49,which is hereby incorporated by reference in its entirety.

In certain embodiments, the DNA-directed library synthesis comprises 3-Dproximity-based library synthesis, also known as YoctoReactortechnology, which is described in, e.g., Blakskjaer et al., Curr OpinChem Biol. (2015) 26:62-7, which is hereby incorporated by reference inits entirety.

In certain embodiments, the method of synthesizing a library of taggedmacrocyclic compounds comprises encoded self-assembling chemical (ESAC)library synthesis, also known as double-pharmacophore DNA-encodedchemical libraries, as described in, e.g., Mannocci et al., Chem.Commun. (2011) 47:12747-53; Melkko et al., Nat. Biotechnol. (2004)22(5):568-74; Scheuermann et al., Bioconjugate Chem. (2008) 19:778-85;and U.S. Pat. No. 8,642,215 to Neri et al. each of which is herebyincorporated by reference in its entirety. In certain embodiments,synthesizing a library of tagged macrocyclic compounds by ESAC synthesiscomprises, for example, non-covalent combinatorial assembly ofcomplementary oligonucleotide sub-libraries, in which each sub-librarycan include a first oligonucleotide appended to a first building block,wherein the first oligonucleotide comprises a coding domain thatidentifies the first building block, and a hybridization domain, whichself-assembles to a second oligonucleotide appended to a second buildingblock, second oligonucleotide comprising a coding domain that identifiesthe second building block, and a hybridization domain thatself-assembles to the first oligonucleotide.

In some embodiments, the method of synthesizing a library of taggedmacrocyclic compounds comprises DNA-routing, as described in, e.g.,Clark, Curr Opin Chem Biol. (2010) 14(3):396-403, which is herebyincorporated by reference in its entirety.

In certain embodiments, oligonucleotide ligation can utilize one ofseveral methods that would be appreciated be a person of ordinary skillin the art, described, for example, in Zimmermann & Neri, Drug Discov.Today. (2016) 21(11):1828-1834; and Keefe et al., Curr Opin Chem Biol.(2015) 26:80-88, each of which are hereby incorporated by reference inits entirety. In certain embodiments, the oligonucleotide ligation canbe an enzymatic ligation. In certain embodiments, the oligonucleotideligation can be a chemical ligation.

In certain embodiments, the ligation comprises base-pairing a short,complementary “adapter” oligonucleotide to single-strandedoligonucleotides to either end of the ligation site, allowing ligationof single-stranded DNA tags in each cycle. See Clark et al., Nat. Chem.Biol. (2009) 5:647-54, which is hereby incorporated by reference in itsentirety. In certain embodiments, the oligonucleotide ligation comprisesutilizing 2-base overhangs at the 3′ end of the headpiece and of eachbuilding block's DNA tag to form sticky ends for ligation. In certainembodiments, the sequences of the overhangs can depend on the cycle butnot on the building block, so that any DNA tag can be ligated to any DNAtag from the previous cycle, but not to a truncated sequence. See id. Incertain embodiments, the oligonucleotide ligation step can utilizeoligonucleotides of opposite sense for subsequent cycles, with a smallregion of overlap in which the two oligonucleotides are complementary.In certain embodiments, in lieu of ligation, DNA polymerase can be usedto fill in the rest of the complementary sequences, creating adouble-strand oligonucleotide comprising both tags. In certainembodiments, the oligonucleotide ligation can be chemical. While notwishing to be bound by theory, it is thought that chemical ligation maypermit greater flexibility with regard to solution conditions and mayreduce the buffer exchange steps necessary. See Keefe et al., Curr OpinChem Biol. (2015) 26:80-88, which is hereby incorporated by reference inits entirety.

In certain embodiments, provided herein is a method for identifying oneor more compounds that bind to a biological target, the methodcomprising: (a) incubating the biological target with at least a portionof a plurality of distinct tagged macrocyclic compounds of a compoundlibrary to make at least one bound compound and at least one unboundcompound of the plurality of distinct tagged macrocyclic compounds; (b)removing the at least one unbound compound; (c) sequencing each of theat least one oligonucleotide (D) of the at least one bound compound. Incertain embodiments, each compound of the plurality of distinct taggedmacrocyclic compounds of the compound library comprises a macrocycliccompound operatively linked to at least one oligonucleotide (D). Incertain embodiments, the macrocyclic compound comprises an FKBD, aneffector domain, a first linking region, and a second linking region. Incertain embodiments, the FKBD, the effector domain, the first linkingregion, and the second linking region together form a macrocycle. Incertain embodiments, each at least one oligonucleotide (D) can identifyat least one of the FKBD, the effector domain, the first linking region,and the second linking region of each of the plurality of distincttagged macrocyclic compounds. In certain embodiments, each compound ofthe plurality of distinct tagged macrocyclic compounds of the compoundlibrary comprises a compound of Formula (A) (as above-defined). Incertain embodiments, each compound of the plurality of distinct taggedmacrocyclic compounds of the compound library comprises a compound ofFormula (I) (as above-defined). As a person of ordinary skill in the artwould be aware, various techniques for synthesizing the library oftagged macrocyclic compounds are described in, e.g., Kuai et al., SLASDiscov. (2018) 23(5):405-416; Brown et al., Annu. Rev. Biochem. (2018)87:5.1-5.24; Roman et al., SLAS Discov. (2018) 23(5):387-396; Amigo etal., SLAS Discov. (2018) 23(5):397-404; Shi et al., Bioconjug Chem.(2017) 20; 28(9):2293-2301; Machutta et al., Nat Commun. (2017) 8:16081;Li et al., Chembiochem. (2017) 4; 18(9):848-852; Satz et al., ACS CombSci. (2017) 10; 19(4):234-238; Denton & Krusemark, MedChemComm, (2016)7(10): 2020-2027; Eidam & Satz, MedChemComm, (2016) 7(7): 1323-1331; Baoet al., Anal. Chem., (2016) 88 (10):5498-5506; Decurtins et al., NatProtoc. (2016) 11(4):764-80; Harris et al., J. Med. Chem. (2016) 59(5):2163-78; Satz, ACS Chem Biol. (2016) 16; 10(10):2237-45; Chan etal., Curr Opin Chem Biol. (2015) 26:55-61; Franzini et al., Chem Commun.(2015) 11; 51(38):8014-16; and Buller et al., Bioorg Med Chem Lett.(2010) 15; 20(14):4188-92, each of which is hereby incorporated byreference in its entirety.

In certain embodiments, the incubating step can be performed underconditions suitable for at least one of the plurality of distinct taggedmacrocyclic compounds of the compound library to bind to the biologicaltarget. A person of ordinary skill in the art would understand whatconditions would be considered suitable for at least one of theplurality of distinct tagged macrocyclic compounds of the compoundlibrary to bind to the biological target.

In certain embodiments, the identifying one or more compounds that bindto a biological target comprises a bind-wash-elute procedure formolecule selection as described in, e.g., Ding et al., ACS Med. Chem.Lett. (2015) 7;6(8):888-93, which is hereby incorporated by reference inits entirety. In certain embodiments, the incubating step (a comprisescontacting the plurality of tagged compounds in the compound librarywith a target protein, wherein the target protein can be immobilized ona substrate (e.g., resin). In certain embodiments, the removing step (b)comprises washing the substrate to remove the at least one unboundcompound. In certain embodiments, the sequencing step (c) comprisessequencing the at least one oligonucleotide (D) to identify which of theplurality of tagged compounds bound to the target protein.

In certain embodiments, the identifying one or more compounds that bindto a biological target comprises utilizing unmodified, non-immobilizedtarget protein. Such methods, which can utilize a aligate-crosslink-purify strategy are described in, e.g., Shi et al.,Bioconjug. Chem. (2017) 20; 28(9):2293-2301, which is herebyincorporated by reference in its entirety. In certain embodiments, othermethods for identifying the one or more compounds that bind to thebiological target can be utilized. Such methods would be apparently to aperson of ordinary skill in the art, and examples of such methods aredescribed in, e.g., Machutta et al., Nat. Commun. (2017) 8:16081; Chanet al., Curr. Opin. Chem. Biol. (2015) 26:55-61; Lim, C&EN, (2017) 95(29):10; Amigo et al., SLAS Discov. (2018) 23(5):397-404; Tian et al.,MedChemComm. (2016) 7(7): 1316-1322; See Satz, CS Comb. Sci. (2016) 18(7):415-424 each of which is hereby incorporated by reference in itsentirety.

Tables 5-7 below show all the Rapafucin molecules in the presentdisclosure, the structural moieties are shown according to Formula (XV)An example of the chemical structure generated from Formula (XV) forcompound 1 is shown below. In the case of amino acid monomers and FKBDs,a dehydration reaction occurs resulting in a peptide bond. Examples thatdo not designate a monomer 4 are Rapafucins composed of an FKBD withlinker and only 3 monomers.

Monomer 1 Monomer 2 Monomer 3 Monomer 4

TABLE 5 Rapafucin compound 1 to compound 578 in this Disclosure. FKBDCompound with Monomer Monomer Monomer Monomer Retention Rel. Prolif.,No. linkers 1 2 3 4 Time A549 1 eFKBD ra147 ra567 ra562 g 4.33 low 2eFKBD ra147 ra566 ra562 g 4.35 low 3 eFKBD ra147 ra58 ra562 g 4.37 low 4eFKBD ra147 ra512 ra562 g 4.32 low 5 eFKBD ra147 ra71 ra562 g 4.19 low 6eFKBD ra147 ra135 ra562 g 4.40 low 7 eFKBD ra147 ra97 ra562 g 4.41 low 8eFKBD ra147 y ra562 g 3.81 low 9 eFKBD ma napA ra562 g 4.71 low 10 eFKBDra147 ra94 ra562 g 4.39 low 11 eFKBD ra147 ra137 ra562 g 4.38 low 12eFKBD ra147 ra98 ra562 g 4.48 low 13 eFKBD ra147 ra73 ra562 g 4.40 low14 eFKBD ra147 ra60 ra562 g 4.43 low 15 eFKBD ra147 ra353 ra562 g 4.53low 16 eFKBD ra147 ra133 ra562 g 3.91 low 17 eFKBD ra147 ra96 ra562 g4.47 low 18 eFKBD ra147 ra95 ra562 g 4.45 low 19 eFKBD ra147 ra70 ra562g 4.48 low 20 eFKBD ra147 ra91 ra562 g 3.51 low 21 eFKBD ra147 ra90ra562 g 3.44 low 22 eFKBD ra147 ra89 ra562 g 3.38 low 23 eFKBD ra147ra301 ra562 g 3.89 low 24 eFKBD ra147 ra68 ra562 g 4.12 low 25 eFKBDra147 ra67 ra562 g 4.13 low 26 eFKBD ra147 ra189 ra562 g 4.11 low 27eFKBD ra147 ra144 ra562 g 4.19 low 28 eFKBD ra147 ra530 ra562 g 4.31 low29 eFKBD ra147 cha ra562 g 4.48 low 30 eFKBD ra147 ra527 ra562 g 4.55low 31 eFKBD ra147 ra549 ra562 g 4.59 low 32 eFKBD ra147 ra59 ra562 g4.66 low 33 eFKBD ra147 tle ra562 g 4.23 low 34 eFKBD ra147 ra83 ra562 g4.31 low 35 eFKBD ra147 ra533 ra562 g 4.39 low 36 eFKBD ra147 ra84 ra562g 4.40 low 37 eFKBD ra147 ra129 ra562 g 4.69 low 38 eFKBD ra147 ra602ra562 g 4.28 low 39 eFKBD ra147 ra122 ra562 g 4.41 low 40 eFKBD ra147ra128 ra562 g 4.29 low 41 eFKBD ra147 ra600 ra562 g 4.29 low 42 eFKBDra147 df ra562 g 4.30 low 43 eFKBD ra147 ra134 ra562 g 4.39 low 44 eFKBDra147 mf ra562 g 4.45 low 45 eFKBD ra147 ra185 ra562 g 4.31 low 46 eFKBDra147 ra124 ra562 g 4.25 low 47 eFKBD ra147 ra113 ra562 g 4.22 low 48eFKBD ra147 ra114 ra562 g 4.17 low 49 eFKBD ra147 ra112 ra562 g 4.14 low50 eFKBD ra147 ra87 ra562 g 4.38 low 51 eFKBD ra147 ra104 ra562 g 4.42low 52 eFKBD ra147 ra63 ra562 g 4.43 low 53 eFKBD ma ra107 ra562 g 4.51medium 54 eFKBD ma ra110 ra209 g 4.22 high 55 eFKBD ra147 ra119 ra562 g4.26 low 56 eFKBD ra147 ra118 ra562 g 4.24 low 57 eFKBD ma ra110 ra562 g4.32 high 58 eFKBD ra147 ra65 ra562 g 4.34 low 59 eFKBD ra147 ra115ra562 g 4.34 low 60 eFKBD ra147 ra117 ra562 g 4.40 low 61 eFKBD ra147ra116 ra562 g 4.35 low 62 eFKBD ra147 ra62 ra562 g 4.49 low 63 eFKBDra147 ra56 ra562 g 4.54 low 64 eFKBD ra147 ra55 ra562 g 4.52 low 65eFKBD ra147 ra366 ra562 g 4.47 low 66 eFKBD ma ra111 ra562 g 3.57 low 67eFKBD ra147 ra109 ra562 g 3.75 low 68 eFKBD ra147 ra525 ra562 g 4.34 low69 eFKBD ra147 ra526 ra562 g 4.37 low 70 eFKBD ra147 ra523 ra562 g 4.93low 71 eFKBD ra147 ra521 ra562 g 4.90 low 72 eFKBD ra147 oic ra562 g4.34 low 73 eFKBD ra147 ra102 ra562 g 4.33 low 74 eFKBD ra147 tic ra562g 4.26 low 75 eFKBD ma ra121 ra562 g 3.96 high 76 eFKBD ra147 ra105ra562 g 4.00 low 77 eFKBD ma ra123 ra562 g 4.47 low 78 eFKBD ma ra567ra562 g 4.58 low 79 eFKBD ma ra566 ra562 g 4.63 low 80 eFKBD ma ra167ra562 g 4.43 low 81 eFKBD ma ra71 ra562 g 4.40 low 82 eFKBD ma ra78ra562 g 4.42 low 83 eFKBD ma ra327 ra562 g 3.66 low 84 eFKBD ma ra324ra562 g 3.62 low 85 eFKBD ma rbphe ra562 g 4.22 low 86 eFKBD ma ra135ra562 g 4.69 low 87 eFKBD ma ra97 ra562 g 4.66 low 88 eFKBD ma y ra562 g3.89 low 89 eFKBD ma ra127 ra562 g 4.21 low 90 eFKBD ma ra171 ra562 g4.33 low 91 eFKBD ma ra175 ra562 g 5.39 low 92 eFKBD ma ra137 ra562 g4.65 low 93 eFKBD ma ra94 ra562 g 4.65 low 94 eFKBD ma ra98 ra562 g 4.86low 95 eFKBD ma ra73 ra562 g 4.70 low 96 eFKBD ma ra60 ra562 g 4.71 low97 eFKBD ma ra353 ra562 g 4.90 low 98 eFKBD ma ra133 ra562 g 3.92 low 99eFKBD ma ra96 ra562 g 4.74 low 100 eFKBD ma ra95 ra562 g 4.73 low 101eFKBD ma ra70 ra562 g 4.74 low 102 eFKBD ma ra491 ra562 g 3.47 low 103eFKBD ma ra91 ra562 g 3.51 low 104 eFKBD ma ra90 ra562 g 3.41 low 105eFKBD ma ra89 ra562 g 3.34 low 106 eFKBD ma ra301 ra562 g 3.90 low 107eFKBD ma ra68 ra562 g 4.19 low 108 eFKBD ma ra67 ra562 g 4.19 low 109eFKBD ma ra347 ra562 g 4.35 low 110 eFKBD ma ra189 ra562 g 4.19 low 111eFKBD ma ra144 ra562 g 4.21 low 112 eFKBD ma ra530 ra562 g 4.40 low 113eFKBD ma ra509 ra562 g 4.52 low 114 eFKBD ma ra507 ra562 g 4.56 low 115eFKBD ma cha ra562 g 4.67 low 116 eFKBD ma ra527 ra562 g 4.72 low 117eFKBD ma ra549 ra562 g 4.88 low 118 eFKBD ma ra59 ra562 g 4.94 low 119eFKBD ma tle ra562 g 4.34 low 120 eFKBD ma ra83 ra562 g 4.40 low 121eFKBD ma ra75 ra562 g 4.53 low 122 eFKBD ma ra533 ra562 g 4.54 low 123eFKBD ma ra84 ra562 g 4.51 low 124 eFKBD ma ra129 ra562 g 4.89 low 125eFKBD ma ra602 ra562 g 4.24 low 126 eFKBD ma ra122 ra562 g 4.41 low 127eFKBD ma ra450 ra562 g 3.95 low 128 eFKBD ma ra522 ra562 g 3.83 low 129eFKBD ma ra128 ra562 g 4.20 low 130 eFKBD ma ra600 ra562 g 4.21 low 131eFKBD ma ra76 ra562 g 4.20 low 132 eFKBD ma df ra562 g 4.34 low 133eFKBD ma ra134 ra562 g 4.41 low 134 eFKBD ma mf ra562 g 4.58 low 135eFKBD ma ra185 ra562 g 4.37 low 136 eFKBD ma ra124 ra562 g 4.34 low 137eFKBD ma ra513 ra562 g 3.99 low 138 eFKBD ma ra113 ra562 g 4.27 low 139eFKBD ma ra114 ra562 g 4.24 low 140 eFKBD ma ra112 ra562 g 4.20 low 141eFKBD ma ra87 ra562 g 4.49 low 142 eFKBD ma ra104 ra562 g 4.50 low 143eFKBD ma ra148 ra562 g 4.13 low 144 eFKBD ma ra63 ra562 g 4.64 low 145eFKBD ma ra561 ra562 g 4.62 low 146 eFKBD ma ra208 ra562 g 4.64 low 147eFKBD ma ra382 ra562 g 4.39 low 148 eFKBD ma ra495 ra562 g 4.64 low 149eFKBD ma ra64 ra562 g 4.46 low 150 eFKBD ma ra119 ra562 g 4.39 low 151eFKBD ma ra118 ra562 g 4.37 low 152 eFKBD ma ra65 ra562 g 4.44 low 153eFKBD ma ra66 ra562 g 4.73 low 154 eFKBD ma ra115 ra562 g 4.49 low 155eFKBD ma ra117 ra562 g 4.55 low 156 eFKBD ma ra116 ra562 g 4.54 low 157eFKBD ma ra62 ra562 g 4.76 low 158 eFKBD ma ra56 ra562 g 4.76 low 159eFKBD ma ra534 ra562 g 4.72 medium 160 eFKBD ma ra88 ra562 g 4.28 low161 eFKBD ma ra55 ra562 g 4.73 low 162 eFKBD ma ra366 ra562 g 4.77 low163 eFKBD ra199 napA ra562 g 4.11 low 164 eFKBD ma ra92 ra562 g 4.56 low165 eFKBD ra202 napA ra562 g 4.17 low 166 eFKBD ra484 napA ra562 g 4.21low 167 eFKBD ma ra93 ra144 g 3.90 medium 168 eFKBD ml ra167 ra562 g4.32 low 169 eFKBD ra207 ra167 ra562 g 4.28 low 170 eFKBD ra565 ra167ra562 g 4.21 low 171 eFKBD ra172 ra167 ra562 g 4.24 low 172 eFKBD ra562ra167 ra562 g 4.33 low 173 eFKBD ra209 ra167 ra562 g 4.28 low 174 eFKBDra61 ra167 ra562 g 4.17 low 175 eFKBD ra74 ra167 ra562 g 4.08 low 176eFKBD ra147 ra332 ra562 g 4.54 low 177 eFKBD ma ra332 ra562 g 4.24 low178 eFKBD ra199 ra332 ra562 g 4.22 low 179 eFKBD ra201 ra332 ra562 g4.30 low 180 eFKBD ra202 ra332 ra562 g 4.30 low 181 eFKBD ra203 ra332ra562 g 4.32 low 182 eFKBD ra484 ra332 ra562 g 4.30 low 183 eFKBD ra379ra332 ra562 g 4.41 low 184 eFKBD ml ra109 ra562 g 3.69 low 185 eFKBDra207 ra109 ra562 g 3.67 low 186 eFKBD ra565 ra109 ra562 g 3.60 low 187eFKBD ra562 ra109 ra562 g 3.72 low 188 eFKBD ra209 ra109 ra562 g 3.71low 189 eFKBD ra61 ra109 ra562 g 3.59 low 190 eFKBD ra74 ra109 ra562 g3.48 low 191 eFKBD ma ra108 ra562 g 3.21 low 192 eFKBD ra199 ra108 ra562g 3.23 low 193 eFKBD ra201 ra108 ra562 g 3.31 low 194 eFKBD ra202 ra108ra562 g 3.33 low 195 eFKBD ra203 ra108 ra562 g 3.36 low 196 eFKBD ra484ra108 ra562 g 3.36 low 197 eFKBD ra379 ra108 ra562 g 3.47 low 198 eFKBDml oic ra562 g 4.25 low 199 eFKBD ra207 oic ra562 g 4.29 low 200 eFKBDra565 oic ra562 g 4.21 low 201 eFKBD ra172 oic ra562 g 4.23 low 202eFKBD ra562 oic ra562 g 4.23 low 203 eFKBD ra209 oic ra562 g 4.27 low204 eFKBD ra61 oic ra562 g 4.14 low 205 eFKBD ra74 oic ra562 g 4.07 low206 eFKBD ra147 ra542 ra562 g 4.25 low 207 eFKBD ma ra542 ra562 g 3.92low 208 eFKBD ra199 ra542 ra562 g 3.91 low 209 eFKBD ra201 ra542 ra562 g4.00 low 210 eFKBD ra202 ra542 ra562 g 3.99 low 211 eFKBD ra203 ra542ra562 g 3.98 low 212 eFKBD ra484 ra542 ra562 g 4.01 low 213 eFKBD ra379ra542 ra562 g 4.13 low 214 eFKBD ml tic ra562 g 4.19 low 215 eFKBD ra207tic ra562 g 4.26 low 216 eFKBD ra565 tic ra562 g 4.14 low 217 eFKBDra172 tic ra562 g 4.16 low 218 eFKBD ra562 tic ra562 g 4.17 low 219eFKBD ra209 tic ra562 g 4.20 low 220 eFKBD ra61 tic ra562 g 4.06 low 221eFKBD ra74 tic ra562 g 4.02 low 222 eFKBD ma ra93 ra209 g 4.06 medium223 eFKBD ma ra136 ra562 g 3.54 low 224 eFKBD ra199 ra136 ra562 g 3.57low 225 eFKBD ra201 ra136 ra562 g 3.62 low 226 eFKBD ra202 ra136 ra562 g3.64 low 227 eFKBD ra203 ra136 ra562 g 3.66 low 228 eFKBD ra484 ra136ra562 g 3.64 low 229 eFKBD ra379 ra136 ra562 g 3.78 low 230 eFKBD mlra545 ra562 g 4.19 low 231 eFKBD ra207 ra545 ra562 g 4.12 low 232 eFKBDra565 ra545 ra562 g 4.10 low 233 eFKBD ra172 ra545 ra562 g 4.11 low 234eFKBD ra562 ra545 ra562 g 4.15 low 235 eFKBD ra209 ra545 ra562 g 4.18low 236 eFKBD ra61 ra545 ra562 g 4.08 medium 237 eFKBD ra74 ra545 ra562g 4.02 low 238 eFKBD ra147 ra350 ra562 g 4.18 low 239 eFKBD ma ra350ra562 g 3.87 low 240 eFKBD ra199 ra350 ra562 g 3.93 low 241 eFKBD ra201ra350 ra562 g 3.96 low 242 eFKBD ra202 ra350 ra562 g 3.97 low 243 eFKBDra203 ra350 ra562 g 3.97 low 244 eFKBD ra484 ra350 ra562 g 4.05 low 245eFKBD ra379 ra350 ra562 g 4.17 low 246 eFKBD ml ra351 ra562 g 4.31 low247 eFKBD ra207 ra351 ra562 g 4.14 low 248 eFKBD ra565 ra351 ra562 g4.16 low 249 eFKBD ra172 ra351 ra562 g 4.19 low 250 eFKBD ra562 ra351ra562 g 4.25 low 251 eFKBD ra209 ra351 ra562 g 4.27 low 252 eFKBD ra61ra351 ra562 g 4.18 low 253 eFKBD ra74 ra351 ra562 g 4.02 low 254 eFKBDma ra93 ra562 g 4.58 low 255 eFKBD ml ra93 ra562 g 4.96 low 256 eFKBDra344 ra102 ra562 g 4.48 low 257 eFKBD ra209 ra102 ra562 g 3.24 low 258eFKBD ra147 ra554 ra562 g 4.96 low 259 eFKBD ma ra554 ra562 g 4.49 low260 eFKBD ra201 ra554 ra562 g 4.57 low 261 eFKBD ra203 ra554 ra562 g4.65 low 262 eFKBD ra344 ra546 ra562 g 4.60 low 263 eFKBD ml ra546 ra562g 4.86 low 264 eFKBD ra565 ra546 ra562 g 4.63 low 265 eFKBD ra209 ra546ra562 g 4.78 low 266 eFKBD ra147 mw ra562 g 4.68 low 267 eFKBD ma mwra562 g 4.37 low 268 eFKBD ra201 mw ra562 g 4.44 low 269 eFKBD ra203 mwra562 g 4.44 low 270 eFKBD ra344 ra354 ra562 g 4.68 low 271 eFKBD mlra354 ra562 g 4.83 low 272 eFKBD ra565 ra354 ra562 g 4.67 low 273 eFKBDra209 ra354 ra562 g 4.80 low 274 eFKBD ra147 ra385 ra562 g 4.89 low 275eFKBD ma ra385 ra562 g 4.45 low 276 eFKBD ra201 ra385 ra562 g 4.54 low277 eFKBD ra203 ra385 ra562 g 4.57 low 278 eFKBD ra344 ra486 ra562 g5.86 low 279 eFKBD ml ra486 ra562 g 5.40 low 280 eFKBD ra565 ra486 ra562g 5.26 low 281 eFKBD ra209 ra486 ra562 g 4.29 low 282 eFKBD ra147 ra487ra562 g 4.34 low 283 eFKBD ma ra487 ra562 g 3.94 low 284 eFKBD ra201ra487 ra562 g 4.03 low 285 eFKBD ra203 ra487 ra562 g 4.07 low 286 eFKBDma ra323 ra562 g 3.20 low 287 eFKBD ra201 ra323 ra562 g 5.30 low 288eFKBD ra203 ra323 ra562 g 5.27 low 289 eFKBD ra344 ra347 ra562 g 4.56low 290 eFKBD ml ra347 ra562 g 4.71 low 291 eFKBD ra565 ra347 ra562 g4.55 low 292 eFKBD ra209 ra347 ra562 g 4.69 low 293 eFKBD ra147 napara209 g 4.29 medium 294 eFKBD ra201 ra88 ra562 g 4.35 low 295 eFKBDra203 ra88 ra562 g 4.39 low 296 eFKBD ra344 ra137 ra562 g 4.90 low 297eFKBD ml ra137 ra562 g 5.06 low 298 eFKBD ra565 ra137 ra562 g 4.89 low299 eFKBD ra209 ra137 ra562 g 5.03 low 300 eFKBD ra147 ra495 ra562 g5.05 low 301 eFKBD ra201 ra495 ra562 g 4.72 low 302 eFKBD ra203 ra495ra562 g 4.76 low 303 eFKBD ra344 ra171 ra562 g 4.53 low 304 eFKBD mlra171 ra562 g 4.69 low 305 eFKBD ra565 ra171 ra562 g 4.53 low 306 eFKBDra209 ra171 ra562 g 4.66 low 307 eFKBD ra201 ra123 ra562 g 4.56 low 308eFKBD ra203 ra123 ra562 g 4.59 low 309 eFKBD ra344 ra93 ra562 g 4.81 low310 eFKBD ra565 ra93 ra562 g 4.77 low 311 eFKBD ra209 ra93 ra562 g 4.91low 312 eFKBD ra147 ra107 ra549 g 4.57 medium 313 eFKBD ra201 ra64 ra562g 4.52 low 314 eFKBD ra203 ra64 ra562 g 4.57 low 315 eFKBD ra344 ra116ra562 g 4.78 low 316 eFKBD ml ra116 ra562 g 4.91 low 317 eFKBD ra565ra116 ra562 g 4.82 low 318 eFKBD ra209 ra116 ra562 g 4.92 low 319 eFKBDra147 ra107 ra562 g 4.44 low 320 eFKBD ra201 ra66 ra562 g 4.82 low 321eFKBD ra203 ra66 ra562 g 4.88 low 322 eFKBD ra344 ra75 ra562 g 4.89 low323 eFKBD ml ra75 ra562 g 5.04 low 324 eFKBD ra565 ra75 ra562 g 4.83 low325 eFKBD ra209 ra75 ra562 g 4.87 low 326 eFKBD ra147 ra108 ra562 g 3.68low 327 eFKBD ra201 ra127 ra562 g 4.31 low 328 eFKBD ra203 ra127 ra562 g4.34 low 329 eFKBD ra344 ra113 ra562 g 4.46 low 330 eFKBD ml ra113 ra562g 4.60 low 331 eFKBD ra565 ra113 ra562 g 4.48 low 332 eFKBD ra209 ra113ra562 g 4.61 low 333 eFKBD ra147 ra497 ra562 g 4.24 low 334 eFKBD ra147ra148 ra562 g 4.39 medium 335 eFKBD ra147 ra110 ra562 g 4.61 medium 336eFKBD ra147 ra111 ra562 g 3.86 low 337 eFKBD ra147 ra121 ra549 g 4.41medium 338 eFKBD ra147 ra121 ra562 g 4.25 low 339 eFKBD ra147 napa ra206g 4.05 medium 340 eFKBD ra147 ra497 ra206 g 3.91 medium 341 eFKBD ra147ra93 ra206 g 4.08 medium 342 eFKBD ra147 ra204 ra206 g 3.91 medium 343eFKBD ra147 ra148 ra206 g 4.06 medium 344 eFKBD ra147 ra121 ra206 g 3.88medium 345 eFKBD ra147 ra107 ra206 g 4.07 medium 346 eFKBD ra147 ra110ra206 g 4.26 medium 347 eFKBD ra147 ra88 ra206 g 3.85 medium 348 eFKBDra147 ra92 ra206 g 4.02 medium 349 eFKBD ra147 ra111 ra206 g 3.61 medium350 eFKBD ra147 ra123 ra562 g 4.28 low 351 eFKBD ra147 ra93 ra209 g 4.33medium 352 eFKBD ra147 ra204 ra209 g 4.17 low 353 eFKBD ra147 ra148ra209 g 4.31 medium 354 eFKBD ra147 ra121 ra209 g 4.17 medium 355 eFKBDra147 ra107 ra209 g 4.33 medium 356 eFKBD ra147 ra110 ra209 g 4.49medium 357 eFKBD ra147 ra88 ra209 g 4.11 low 358 eFKBD ra147 ra92 ra209g 4.28 medium 359 eFKBD ra147 ra111 ra209 g 3.86 low 360 eFKBD ra147napa ra106 g 4.16 low 361 eFKBD ra147 ra497 ra106 g 4.06 low 362 eFKBDra147 ra93 ra106 g 4.20 low 363 eFKBD ra147 ra204 ra106 g 4.06 low 364eFKBD ra147 ra148 ra106 g 4.17 low 365 eFKBD ra147 ra121 ra106 g 4.02low 366 eFKBD ra147 ra107 ra106 g 4.19 low 367 eFKBD ra147 ra110 ra106 g4.36 low 368 eFKBD ra147 ra88 ra106 g 3.97 low 369 eFKBD ra147 ra92ra106 g 4.15 low 370 eFKBD ra147 ra111 ra106 g 3.74 low 371 eFKBD ra147napa ra189 g 4.17 low 372 eFKBD ra147 ra497 ra189 g 4.06 low 373 eFKBDra147 ra93 ra189 g 4.20 low 374 eFKBD ra147 ra204 ra189 g 4.06 low 375eFKBD ra147 ra148 ra189 g 4.18 low 376 eFKBD ra147 ra121 ra189 g 4.02low 377 eFKBD ra147 ra107 ra189 g 4.20 low 378 eFKBD ra147 ra110 ra189 g4.36 low 379 eFKBD ra147 ra88 ra189 g 3.98 low 380 eFKBD ra147 ra92ra189 g 4.16 low 381 eFKBD ra147 ra111 ra189 g 3.74 low 382 eFKBD ra147napa ra144 g 4.17 low 383 eFKBD ra147 ra497 ra144 g 4.03 low 384 eFKBDra147 ra93 ra144 g 4.19 low 385 eFKBD ra147 ra204 ra144 g 4.07 low 386eFKBD ra147 ra121 ra144 g 4.03 medium 387 eFKBD ra147 ra107 ra144 g 4.19low 388 eFKBD ra147 ra110 ra144 g 4.39 medium 389 eFKBD ra147 ra88 ra144g 3.96 low 390 eFKBD ra147 ra92 ra144 g 4.16 medium 391 eFKBD ra147ra111 ra144 g 3.73 low 392 eFKBD ra147 napa ra126 g 4.00 low 393 eFKBDra147 ra497 ra126 g 3.84 low 394 eFKBD ra147 ra93 ra126 g 4.03 low 395eFKBD ra147 ra511 ra126 g 4.03 low 396 eFKBD ra147 ra204 ra126 g 3.87low 397 eFKBD ra147 ra148 ra126 g 4.00 low 398 eFKBD ra147 ra121 ra126 g3.83 low 399 eFKBD ra147 ra107 ra126 g 4.03 low 400 eFKBD ra147 ra110ra126 g 4.18 low 401 eFKBD ra147 ra88 ra126 g 3.77 low 402 eFKBD ra147ra92 ra126 g 3.98 low 403 eFKBD ra147 ra111 ra126 g 3.51 low 404 eFKBDra147 napa ra549 g 4.54 low 405 eFKBD ra147 ra127 ra562 g 4.22 low 406eFKBD ra147 ra93 ra549 g 4.58 low 407 eFKBD ra147 ra204 ra549 g 4.43 low408 eFKBD ra147 ra148 ra549 g 4.55 medium 409 eFKBD ra147 ra136 ra562 g4.00 low 410 eFKBD ra147 ra110 ra549 g 4.78 medium 411 eFKBD ra147 ra88ra549 g 4.34 medium 412 eFKBD ra147 ra92 ra549 g 4.53 medium 413 eFKBDra147 ra111 ra549 g 4.15 low 414 eFKBD ma ra497 ra562 g 3.94 low 415eFKBD ra147 ra148 ra144 g 4.18 medium 416 eFKBD ra147 ra497 ra209 g 4.17medium 417 eFKBD ra147 ra497 ra549 g 4.43 medium 418 eFKBD ra147 ra64ra562 g 4.32 low 419 eFKBD ma ra497 ra206 g 3.57 low 420 eFKBD ma ra93ra206 g 3.80 low 421 eFKBD ma ra204 ra206 g 3.57 low 422 eFKBD ma ra148ra206 g 3.74 low 423 eFKBD ma ra121 ra206 g 3.53 low 424 eFKBD ma ra107ra206 g 3.79 low 425 eFKBD ma ra110 ra206 g 3.96 low 426 eFKBD ma ra88ra206 g 3.49 low 427 eFKBD ma ra92 ra206 g 3.72 low 428 eFKBD ma napara209 g 4.04 low 429 eFKBD ma ra497 ra209 g 3.91 medium 430 eFKBD mara204 ra209 g 3.91 low 431 eFKBD ma ra148 ra209 g 4.04 low 432 eFKBD mara107 ra209 g 4.06 low 433 eFKBD ra147 ra66 ra562 g 4.49 low 434 eFKBDma ra88 ra209 g 3.83 medium 435 eFKBD ma napa ra106 g 3.90 low 436 eFKBDma ra497 ra106 g 3.75 low 437 eFKBD ma ra93 ra106 g 3.93 low 438 eFKBDma ra204 ra106 g 3.74 low 439 eFKBD ma ra148 ra106 g 3.91 low 440 eFKBDma ra121 ra106 g 3.72 low 441 eFKBD ma ra107 ra106 g 3.93 low 442 eFKBDma ra110 ra106 g 4.10 low 443 eFKBD ma ra88 ra106 g 3.66 low 444 eFKBDma ra92 ra106 g 3.90 low 445 eFKBD ma ra111 ra106 g 3.35 low 446 eFKBDma napa ra189 g 3.86 low 447 eFKBD ma ra497 ra189 g 3.71 low 448 eFKBDma ra93 ra189 g 3.90 low 449 eFKBD ma ra204 ra189 g 3.72 low 450 eFKBDma ra148 ra189 g 3.86 low 451 eFKBD ma ra121 ra189 g 3.67 low 452 eFKBDma ra107 ra189 g 3.90 low 453 eFKBD ma ra110 ra189 g 4.07 low 454 eFKBDma ra88 ra189 g 3.65 low 455 eFKBD ma ra92 ra189 g 3.85 low 456 eFKBD mara111 ra189 g 3.33 low 457 eFKBD ma napa ra144 g 3.87 low 458 eFKBD mara497 ra144 g 3.70 medium 459 eFKBD ma ra204 ra144 g 3.69 low 460 eFKBDma ra148 ra144 g 3.88 low 461 eFKBD ma ra121 ra144 g 3.70 medium 462eFKBD ma ra107 ra144 g 3.91 low 463 eFKBD ma ra110 ra144 g 4.08 medium464 eFKBD ma ra88 ra144 g 3.63 low 465 eFKBD ma ra92 ra144 g 3.87 low466 eFKBD ma ra111 ra144 g 3.30 low 467 eFKBD ma ra497 ra126 g 3.46 low468 eFKBD ma ra148 ra126 g 3.67 low 469 eFKBD ma ra121 ra126 g 3.44 low470 eFKBD ma ra107 ra126 g 3.72 low 471 eFKBD ma ra110 ra126 g 3.89 low472 eFKBD ma ra92 ra126 g 3.69 low 473 eFKBD ma ra111 ra126 g 3.04 low474 eFKBD ma napa ra549 g 4.29 low 475 eFKBD ma ra497 ra549 g 4.16 low476 eFKBD ma ra93 ra549 g 4.31 low 477 eFKBD ma ra204 ra549 g 4.14 low478 eFKBD ma ra148 ra549 g 4.31 low 479 eFKBD ma ra121 ra549 g 4.15 low480 eFKBD ma ra107 ra549 g 4.32 low 481 eFKBD ma ra110 ra549 g 4.51 low482 eFKBD ma ra88 ra549 g 4.09 low 483 eFKBD ma ra92 ra549 g 4.29 low484 eFKBD ma ra111 ra549 g 3.86 low 485 eFKBD ra147 ra88 ra562 g 4.13low 486 eFKBD ml napa ra549 g 5.13 low 487 eFKBD ml napa ra144 g 4.53low 488 eFKBD mi napa ra562 g 4.85 low 489 eFKBD mi napa ra549 g 5.10low 490 eFKBD mv napa ra209 g 4.56 low 491 eFKBD ra379 napa ra549 g 4.96low 492 eFKBD ra379 napa ra144 g 4.40 low 493 eFKBD ra203 ra185 ra209 g4.31 low 494 eFKBD ra202 ra185 ra209 g 4.48 low 495 eFKBD ra310 ra185ra209 g 4.68 low 496 eFKBD ra203 ra110 ra562 g 4.95 low 497 eFKBD ra202ra110 ra562 g 4.91 low 498 eFKBD ra310 ra110 ra562 g 5.32 low 499 eFKBDra203 ra93 ra209 g 4.46 low 500 eFKBD ra202 ra93 ra209 g 4.47 low 501eFKBD ra310 ra93 ra209 g 4.80 low 502 eFKBD ra147 ra92 ra562 g 4.41 low503 eFKBD mi ra497 ra209 g 4.54 low 504 eFKBD mi ra497 ra549 g 4.93 low505 eFKBD mi ra497 ra144 g 4.32 low 506 eFKBD ra379 ra497 ra562 g 4.48low 507 eFKBD ra379 ra497 ra209 g 4.40 low 508 eFKBD ra379 ra497 ra549 g4.78 low 509 eFKBD ra379 ra497 ra144 g 4.20 low 510 eFKBD ra147 ra93ra562 g 4.44 low 511 eFKBD ml ra93 ra549 g 5.05 low 512 eFKBD ra201 napAra562 g 4.15 low 513 eFKBD mi ra93 ra562 g 4.83 low 514 eFKBD mi ra93ra209 g 4.66 low 515 eFKBD mi ra93 ra549 g 5.06 low 516 eFKBD mi ra93ra144 g 4.47 low 517 eFKBD ra379 ra93 ra562 g 4.70 low 518 eFKBD ra379ra93 ra209 g 4.56 low 519 eFKBD ra379 ra93 ra549 g 4.91 low 520 eFKBDra379 ra93 ra144 g 4.37 low 521 eFKBD ml ra148 ra562 g 4.91 low 522eFKBD ml ra148 ra209 g 4.73 low 523 eFKBD ml ra148 ra549 g 5.17 low 524eFKBD mi ra148 ra562 g 4.86 low 525 eFKBD mi ra148 ra209 g 4.69 low 526eFKBD mi ra148 ra549 g 5.12 low 527 eFKBD mi ra148 ra144 g 4.51 low 528eFKBD ra379 ra148 ra562 g 4.74 low 529 eFKBD ra379 ra148 ra209 g 4.59low 530 eFKBD ra379 ra148 ra549 g 4.98 low 531 eFKBD ra379 ra148 ra144 g4.40 low 532 eFKBD ra203 napA ra562 g 4.18 low 533 eFKBD ml ra107 ra209g 4.72 low 534 eFKBD ml ra107 ra144 g 4.52 low 535 eFKBD mi ra107 ra562g 4.83 low 536 eFKBD mi ra107 ra209 g 4.69 low 537 eFKBD mi ra107 ra549g 5.09 low 538 eFKBD mi ra107 ra144 g 4.49 low 539 eFKBD ra379 ra107ra562 g 4.73 low 540 eFKBD ra379 ra107 ra209 g 4.57 low 541 eFKBD ra379ra107 ra549 g 4.94 low 542 eFKBD ra379 ra107 ra144 g 4.40 low 543 eFKBDml ra121 ra562 g 4.64 low 544 eFKBD ml ra121 ra209 g 4.49 low 545 eFKBDra379 napA ra562 g 4.30 low 546 eFKBD ml ra121 ra144 g 4.33 low 547eFKBD mi ra121 ra562 g 4.60 low 548 eFKBD mi ra121 ra209 g 4.48 low 549eFKBD mi ra121 ra549 g 4.86 low 550 eFKBD mi ra121 ra144 g 4.30 low 551eFKBD ra379 ra121 ra562 g 4.48 low 552 eFKBD ra379 ra121 ra209 g 4.35low 553 eFKBD ra379 ra121 ra549 g 4.71 low 554 eFKBD ra379 ra121 ra144 g4.18 low 555 eFKBD ra347 ra110 ra144 g 4.98 low 556 eFKBD ra319 ra110ra562 g 4.78 low 557 eFKBD ra319 ra110 ra209 g 4.59 low 558 eFKBD ra319ra110 ra549 g 4.94 low 559 eFKBD ra319 ra110 ra144 g 4.44 low 560 rae1ra147 napA ra562 g 5.56 medium 561 rae2 ra147 napA ra562 g 5.63 medium562 rae3 ra147 napA ra562 g 5.48 medium 563 rae4 ra147 napA ra562 g 5.47low 564 rae5 ra147 napA ra562 g 5.48 low 565 rae9 ra147 napA ra562 g5.35 medium 566 rae10 ra147 napA ra562 g 5.10 medium 567 rae11 ra147napA ra562 g 5.11 medium 568 rae12 ra147 napA ra562 g 5.74 medium 569rae13 ra147 napA ra562 g 5.27 medium 570 rae14 ra147 napA ra562 g 5.72medium 571 rae16 ra147 napA ra562 g 5.93 low 572 rae17 ra147 napA ra562g 4.41 medium 573 rae18 ra147 napA ra562 g 5.49 low 574 rae19 ra147 napAra562 g 5.60 low 575 eFKBD ra147 napA ra562 g 5.44 low 576 rae20 ra147napA ra562 g 5.56 medium 577 eFKBD 2-Nal mSerBu Gly 6.45 low 578 eFKBD2-Nal mNle Gly 6.44 low

TABLE 6 Rapafucin compound 579 to compound 877. FKBD Compound withMonomer Monomer Monomer Monomer Retention Rel. Prolif., No. linkers 1 23 4 Time NCI-H929 579 eFKBD mf dF sar dF 4.105 low 580 eFKBD ra208 dFsar dF 4.158 low 581 eFKBD ra561 dF sar dF 4.189 low 582 eFKBD ra531 dFsar dF 4.252 low 583 eFKBD ra382 dF sar dF 4.055 low 584 eFKBD ra537 dFsar dF 4.042 low 585 eFKBD ra577 dF sar dF 3.342 low 586 eFKBD ra450 dFsar dF 3.767 low 587 eFKBD ra522 dF sar dF 3.671 low 588 eFKBD ra513 dFsar dF 3.769 low 589 eFKBD ra509 dF sar dF 4.171 low 590 eFKBD ra507 dFsar dF 4.143 low 591 eFKBD ra534 dF sar dF 4.221 low 592 eFKBD ra578 dFsar dF 3.71 low 593 eFKBD ra523 dF sar dF 3.198 low 594 eFKBD ra521 dFsar dF 3.308 low 595 eFKBD ra520 dF sar dF 3.646 low 596 eFKBD ra549 dFsar dF 4.392 low 597 eFKBD ra600 dF sar dF 3.969 low 598 eFKBD ra551 dFsar dF 4.233 low 599 eFKBD ra518 dF sar dF 3.876 low 600 eFKBD cha dFsar dF 4.264 high 601 eFKBD ra527 dF sar dF 4.257 high 602 eFKBD ra566dF sar dF 4.215 low 603 eFKBD ra567 dF sar dF 4.189 low 604 eFKBD ra533dF sar dF 4.135 low 605 eFKBD ra530 dF sar dF 4.111 low 606 eFKBD ra579dF sar dF 3.649 low 607 eFKBD ra55 dF sar dF 4.26 low 608 eFKBD ra56 dFsar dF 4.259 low 609 eFKBD tza dF sar dF 3.759 low 610 eFKBD ra58 dF sardF 3.607 low 611 eFKBD ra59 dF sar dF 4.367 low 612 eFKBD ra60 dF sar dF5.05 low 613 eFKBD ra61 dF sar dF 4.001 low 614 eFKBD ra62 dF sar dF4.283 low 615 eFKBD ra63 dF sar dF 4.23 low 616 eFKBD ra64 dF sar dF4.87 low 617 eFKBD ra65 dF sar dF 4.156 low 618 eFKBD ra66 dF sar dF4.303 low 619 eFKBD ra67 dF sar dF 3.968 low 620 eFKBD ra68 dF sar dF3.983 low 621 eFKBD ra69 dF sar dF 4.076 low 622 eFKBD ra70 dF sar dF4.286 low 623 eFKBD ra71 dF sar dF 4.111 low 624 eFKBD ra73 dF sar dF4.283 low 625 eFKBD ra74 dF sar dF 3.899 low 626 eFKBD ra75 dF sar dF4.16 low 627 eFKBD ra76 dF sar dF 4.616 low 628 eFKBD ra511 dF sar dF4.289 low 629 eFKBD ra78 dF sar dF 4.119 low 630 eFKBD ra79 dF sar dF4.255 low 631 eFKBD ra83 dF sar dF 4.065 low 632 eFKBD ra84 dF sar dF4.155 low 633 eFKBD ra87 dF sar dF 4.123 low 634 eFKBD ra88 dF sar dF4.023 low 635 eFKBD ra89 dF sar dF 3.242 low 636 eFKBD ra90 dF sar dF3.298 low 637 eFKBD ra91 dF sar dF 3.418 low 638 eFKBD ra92 dF sar dF4.206 low 639 eFKBD ra93 dF sar dF 4.232 low 640 eFKBD ra94 dF sar dF4.245 low 641 eFKBD ra95 dF sar dF 4.3 low 642 eFKBD ra96 dF sar dF 4.3low 643 eFKBD ra97 dF sar dF 4.231 low 644 eFKBD ra98 dF sar dF 4.33 low645 eFKBD ra353 dF sar dF 4.358 low 646 eFKBD ra104 dF sar dF 4.133 low647 eFKBD ra106 dF sar dF 3.942 low 648 eFKBD ra107 dF sar dF 4.228 low649 eFKBD ra108 dF sar dF 3.467 low 650 eFKBD ra110 dF sar dF 4.368 low651 eFKBD ra111 dF sar dF 3.74 low 652 eFKBD ra112 dF sar dF 3.984 low653 eFKBD ra113 dF sar dF 4.007 low 654 eFKBD ra114 dF sar dF 3.994 low655 eFKBD ra115 dF sar dF 4.161 low 656 eFKBD ra116 dF sar dF 4.194 low657 eFKBD ra117 dF sar dF 4.201 low 658 eFKBD ra119 dF sar dF 4.101 low659 eFKBD ra120 dF sar dF 4.164 low 660 eFKBD ra121 dF sar dF 4.091 low661 eFKBD ra123 dF sar dF 1.825 low 662 eFKBD ra124 dF sar dF 4.07 low663 eFKBD ra126 dF sar dF 3.797 low 664 eFKBD ra127 dF sar dF 4.014 low665 eFKBD ra128 dF sar dF 4.012 low 666 eFKBD ra132 dF sar dF 3.863 low667 eFKBD ra135 dF sar dF 4.226 low 668 eFKBD ra144 dF sar dF 4.573 low669 eFKBD ra148 dF sar dF 4.164 low 670 eFKBD ra171 dF sar dF 4.013 low671 eFKBD ra173 dF sar dF 3.614 low 672 eFKBD ra175 dF sar dF 4.582 low673 eFKBD ra176 dF sar dF 3.334 medium 674 eFKBD ra185 dF sar dF 4.055low 675 eFKBD mf ra537 sar dF 4.129 low 676 eFKBD ra561 ra537 sar dF4.139 low 677 eFKBD ra63 ra537 sar dF 4.182 low 678 eFKBD ra526 ra537sar dF 4.129 low 679 eFKBD cha ra537 sar dF 4.239 low 680 eFKBD ra75ra537 sar dF 4.145 low 681 eFKBD mf ra507 sar dF 4.218 low 682 eFKBDra521 ra507 sar dF 3.257 low 683 eFKBD ra347 ra507 sar dF 4.142 low 684eFKBD ra354 ra507 sar dF 4.188 low 685 eFKBD ra64 ra507 sar dF 4.202 low686 eFKBD ra89 ra507 sar dF 0.393 low 687 eFKBD mf ra521 sar dF 3.353medium 688 eFKBD ra561 ra521 sar dF 3.51 low 689 eFKBD ra382 ra521 sardF 3.329 low 690 eFKBD ra513 ra521 sar dF 3.096 low 691 eFKBD ra75 ra521sar dF 3.423 low 692 eFKBD tza ra521 sar dF 2.97 low 693 eFKBD mf ra527sar dF 4.32 low 694 eFKBD napa ra527 sar dF 4.386 low 695 eFKBD chara527 sar dF 4.496 low 696 eFKBD ra107 ra527 sar dF 4.399 low 697 eFKBDra63 ra527 sar dF 4.425 low 698 eFKBD ra171 ra527 sar dF 4.191 low 699eFKBD mf ra566 sar dF 4.256 low 700 eFKBD ra521 ra566 sar dF 3.42 low701 eFKBD ra347 ra566 sar dF 4.179 low 702 eFKBD ra107 ra566 sar dF4.331 low 703 eFKBD ra64 ra566 sar dF 4.102 low 704 eFKBD tza ra566 sardF 3.929 low 705 eFKBD mf napa sar dF 4.254 low 706 eFKBD napa napa sardF 4.311 low 707 eFKBD cha napa sar dF 4.383 low 708 eFKBD ra354 napasar dF 4.232 low 709 eFKBD ra171 napa sar dF 4.167 low 710 eFKBD ra89napa sar dF 3.46 low 711 eFKBD mf ra55 sar dF 4.326 low 712 eFKBD ra561ra55 sar dF 4.363 low 713 eFKBD ra526 ra55 sar dF 4.283 low 714 eFKBDra63 ra55 sar dF 4.37 low 715 eFKBD ra171 ra55 sar dF 4.159 low 716eFKBD ra89 ra55 sar dF 3.451 low 717 eFKBD mf ra56 sar dF 4.261 low 718eFKBD ra561 ra56 sar dF 4.343 low 719 eFKBD ra513 ra56 sar dF 3.919 low720 eFKBD ra347 ra56 sar dF 4.202 low 721 eFKBD ra75 ra56 sar dF 4.305low 722 eFKBD ra173 ra56 sar dF 3.822 low 723 eFKBD mf ra59 sar dF 4.381low 724 eFKBD ra526 ra59 sar dF 4.353 low 725 eFKBD cha ra59 sar dF4.598 low 726 eFKBD ra107 ra59 sar dF 4.514 low 727 eFKBD ra75 ra59 sardF 4.487 low 728 eFKBD tza ra59 sar dF 4.06 low 729 eFKBD mf ra60 sar dF4.373 low 730 eFKBD napa ra60 sar dF 4.444 low 731 eFKBD ra382 ra60 sardF 4.338 low 732 eFKBD ra107 ra60 sar dF 4.46 low 733 eFKBD ra64 ra60sar dF 4.358 low 734 eFKBD ra89 ra60 sar dF 3.661 low 735 eFKBD mf ra65sar dF 4.229 low 736 eFKBD ra561 ra65 sar dF 4.288 low 737 eFKBD ra347ra65 sar dF 4.142 low 738 eFKBD ra354 ra65 sar dF 4.185 low 739 eFKBDra171 ra65 sar dF 4.12 low 740 eFKBD ra173 ra65 sar dF 3.776 low 741eFKBD mf ra67 sar dF 4.046 low 742 eFKBD napa ra67 sar dF 4.144 low 743eFKBD ra513 ra67 sar dF 3.696 low 744 eFKBD ra382 ra67 sar dF 4.009 low745 eFKBD ra171 ra67 sar dF 3.991 low 746 eFKBD ra173 ra67 sar dF 3.56low 747 eFKBD mf ra70 sar dF 4.417 low 748 eFKBD ra513 ra70 sar dF 4.104low 749 eFKBD ra63 ra70 sar dF 4.504 low 750 eFKBD ra107 ra70 sar dF4.477 low 751 eFKBD ra75 ra70 sar dF 4.461 low 752 eFKBD ra354 ra70 sardF 4.461 low 753 eFKBD mf ra144 sar dF 4.082 low 754 eFKBD napa ra144sar dF 4.215 low 755 eFKBD ra173 ra144 sar dF 3.611 low 756 eFKBD chara144 sar dF 4.216 low 757 eFKBD ra354 ra144 sar dF 4.111 low 758 eFKBDmf ra354 sar dF 4.315 low 759 eFKBD ra513 ra354 sar dF 3.942 low 760eFKBD ra382 ra354 sar dF 4.351 low 761 eFKBD ra64 ra354 sar dF 4.354 low762 eFKBD ra63 ra354 sar dF 4.485 low 763 eFKBD ra89 ra354 sar dF 3.554low 764 eFKBD mf ra533 sar dF 4.273 low 765 eFKBD ra347 ra533 sar dF4.204 low 766 eFKBD ra382 ra533 sar dF 4.252 low 767 eFKBD ra173 ra533sar dF 3.845 low 768 eFKBD ra64 ra533 sar dF 4.325 low 769 eFKBD mfra567 sar ra60 5.28 low 770 eFKBD mf ra537 sar ra525 4.74 low 771 eFKBDmf ra527 sar ra537 4.993 low 772 eFKBD mf ra537 sar ra566 4.871 low 773eFKBD mf ra567 sar ra537 4.881 low 774 eFKBD mf ra537 sar ra533 4.765low 775 eFKBD mf ra59 sar ra537 5.226 low 776 eFKBD mf ra537 sar ra604.989 low 777 eFKBD mf ra537 sar ra67 4.5 low 778 eFKBD mf ra70 sarra537 5.023 low 779 eFKBD mf ra537 sar ra144 4.505 low 780 eFKBD mfra354 sar ra537 4.749 low 781 eFKBD mf ra507 sar ra525 4.948 low 782eFKBD mf ra507 sar ra566 5.088 low 783 eFKBD mf ra567 sar ra507 5.034low 784 eFKBD mf ra507 sar ra533 4.97 low 785 eFKBD mf ra55 sar ra5075.175 low 786 eFKBD mf ra507 sar ra56 5.191 low 787 eflcbd mf ra59 sarra507 5.424 low 788 eFKBD mf ra507 sar ra60 5.184 low 789 eFKBD mf ra65sar ra507 4.886 low 790 eFKBD mf ra67 sar ra507 4.656 low 791 eFKBD mfra70 sar ra507 5.206 low 792 eFKBD mf ra507 sar ra144 4.666 low 793eFKBD mf ra354 sar ra507 4.898 low 794 eFKBD mf ra566 sar ra521 3.993low 795 eFKBD mf ra533 sar ra525 4.247 low 796 eFKBD mf ra56 sar ra5214.04 low 797 eFKBD mf ra60 sar ra537 5.01 low 798 eFKBD mf ra67 sarra537 4.523 low 799 eFKBD mf ra537 sar ra70 4.998 low 800 eFKBD mf ra144sar ra537 4.516 low 801 eFKBD mf ra537 sar ra354 4.732 low 802 eFKBD mfra566 sar ra527 5.259 low 803 eFKBD mf ra527 sar ra567 5.237 low 804eFKBD mf ra527 sar ra55 5.356 low 805 eFKBD mf ra56 sar ra527 5.375 low806 eFKBD mf ra527 sar ra59 5.647 low 807 eFKBD mf ra60 sar ra527 5.345low 808 eFKBD mf ra527 sar ra65 5.033 low 809 eFKBD mf ra67 sar ra5274.798 low 810 eFKBD mf ra70 sar ra533 5.155 low 811 eFKBD mf ra527 sarra354 5.076 low 812 eFKBD mf ra567 sar ra566 5.11 low 813 eFKBD mf ra59sar ra566 5.479 low 814 eFKBD mf ra566 sar ra60 5.242 low 815 eFKBD mfra65 sar ra566 4.932 low 816 eFKBD mf ra566 sar ra67 4.716 low 817 eFKBDmf ra70 sar ra566 5.298 low 818 eFKBD mf ra566 sar ra144 4.729 low 819eFKBD mf ra354 sar ra566 4.968 low 820 eFKBD mf ra566 sar ra533 5.027low 821 eFKBD mf ra59 sar ra567 5.461 low 822 eFKBD mf ra65 sar ra5674.938 low 823 eFKBD mf ra567 sar ra67 4.706 low 824 eFKBD mf ra70 sarra567 5.267 low 825 eFKBD mf ra55 sar ra533 5.146 low 826 eFKBD mf ra59sar ra533 5.378 low 827 eFKBD mf ra533 sar ra60 5.166 low 828 eFKBD mfra65 sar ra533 4.851 low 829 eFKBD mf ra533 sar ra67 4.65 low 830 eFKBDmf ra533 sar ra144 4.659 low 831 eFKBD mf ra354 sar ra533 4.889 low 832eFKBD mf ra59 sar ra55 5.603 low 833 eFKBD mf ra55 sar ra60 5.352 low834 eFKBD mf ra65 sar ra55 5.028 low 835 eFKBD mf ra67 sar ra55 4.798low 836 eFKBD mf ra70 sar ra55 5.382 low 837 eFKBD mf ra55 sar ra1444.811 low 838 eFKBD mf ra59 sar ra56 5.631 low 839 eFKBD mf ra56 sarra60 5.367 low 840 eFKBD mf ra65 sar ra56 5.049 low 841 eFKBD mf ra56sar ra67 4.82 low 842 eFKBD mf ra70 sar ra56 5.411 low 843 eFKBD mfra354 sar ra56 5.079 low 844 eFKBD mf ra59 sar ra60 5.553 low 845 eFKBDmf ra65 sar ra59 5.23 low 846 eFKBD mf ra70 sar ra59 5.602 low 847 eFKBDmf ra59 sar ra144 4.976 low 848 eFKBD mf ra354 sar ra59 5.25 low 849eFKBD mf ra60 sar ra65 5.031 low 850 eFKBD mf ra67 sar ra60 4.813 low851 eFKBD mf ra60 sar ra70 5.349 low 852 eFKBD mf ra67 sar ra65 4.54 low853 eFKBD mf ra65 sar ra70 5.053 low 854 eFKBD mf ra144 sar ra65 4.54low 855 eFKBD mf ra65 sar ra354 4.771 low 856 eFKBD mf ra144 sar ra554.77 low 857 eFKBD mf ra354 sar ra55 5.049 low 858 eFKBD mf ra70 sarra144 4.834 low 859 eFKBD mf ra354 sar ra70 5.081 low 860 eFKBD mf ra144sar ra354 4.574 low 861 eFKBD mf ra527 sar ra507 5.191 low 862 efkbdra606 df sar df 5.285 high 863 rae21 ra98 df sar df 4.281 low 864 rae19ra98 df sar df 4.22 low 865 aFKBD ra98 df sar df 4.098 low 866 eflcbdra607 df sar df 5.077 high 867 rae21 ra492 df sar df 5.75 low 868 rae19ra492 df sar df 5.54 low 869 aFKBD ra492 df sar df 5.403 low 870 efkbdra608 df sar df 4.948 low 871 rae34 mf df sar df 3.854 low 872 rae35 mfdf sar df 4.434 low 873 raa19 mf df sar df 4.871 low 874 raa20 mf df sardf 4.622 low 875 rae36 mf df sar df 5.43 low 876 rae27 mf df sar df4.962 low 877 rae37 ra398 df sar df 4.181 kmv

TABLE 7 Rapafucin compound 878 to compound 1604. FKBD Rel. Compound withMonomer Monomer Monomer Monomer Retention Uptake, No. linkers 1 2 3 4Time 293T 878 aFKBD ra104 mf dp ml 5.14 low 879 aFKBD ml p ra195 f 4.22low 880 aFKBD ml p mf f 4.24 low 881 aFKBD ml dp ra195 f 4.33 low 882aFKBD ra207 p ra195 f 4.33 low 883 aFKBD ml dp mf f 4.33 low 884 aFKBDra207 p mf f 4.16 low 885 aFKBD ra207 dp ra195 f 4.10 low 886 aFKBD fra195 p ml 4.14 low 887 aFKBD f ra195 p ra207 4.18 low 888 aFKBD f ra195dp ml 4.13 low 889 aFKBD f mf P ml 4.05 low 890 aFKBD dF ra195 p ml 4.06low 891 aFKBD f mf dp ml 4.14 low 892 aFKBD dF ra195 dp ml 4.11 low 893aFKBD dF mf p ml 4.11 low 894 aFKBD ra381 mf dp ml 4.15 low 895 aFKBDra400 mf dp ml 4.13 medium 896 aFKBD ra329 mf dp ml 4.10 medium 897aFKBD ra325 mf dp ml 4.17 medium 898 aFKBD ra516 mf dp ml 4.27 high 899aFKBD ra381 f dp ml 4.06 low 900 aFKBD ra400 f dp ml 4.06 low 901 aFKBDra329 f dp ml 4.03 low 902 aFKBD ra325 f dp ml 4.11 low 903 aFKBD ra516f dp ml 4.17 high 904 aFKBD ra522 f dp ml 3.78 low 905 aFKBD ra450 f dpml 3.89 high 906 aFKBD ra602 f dp ml 4.04 high 907 aFKBD ra381 dF dp ml4.07 medium 908 aFKBD ra400 dF dp ml 4.08 low 909 aFKBD ra329 dF dp ml4.05 medium 910 aFKBD ra325 dF dp ml 4.18 medium 911 aFKBD ra516 dF dpml 4.29 low 912 aFKBD ra522 dF dp ml 3.87 low 913 aFKBD ra450 dF dp ml3.93 low 914 aFKBD ra602 dF dp ml 4.11 low 915 aFKBD ra381 ra195 dp ml4.10 low 916 aFKBD ra400 ra195 dp ml 4.12 low 917 aFKBD ra329 ra195 dpml 4.08 low 918 aFKBD ra325 ra195 dp ml 4.18 low 919 aFKBD ra516 ra195dp ml 4.26 low 920 aFKBD ra522 ra195 dp ml 3.82 low 921 aFKBD ra450ra195 dp ml 3.91 low 922 aFKBD ra602 ra195 dp ml 4.11 low 923 aFKBDra381 y dp ml 3.79 low 924 aFKBD ra400 y dp ml 3.78 low 925 aFKBD ra329y dp ml 3.76 low 926 aFKBD ra325 y dp ml 3.82 low 927 aFKBD ra516 y dpml 3.89 high 928 aFKBD ra602 ra577 dp ml 3.45 low 929 aFKBD ra602 ra173dp ml 3.60 low 930 aFKBD ra602 ra66 dp ml 4.29 medium 931 aFKBD ra602ra56 dp ml 4.30 low 932 aFKBD ra602 ra64 dp ml 4.13 high 933 aFKBD ra602ra171 dp ml 4.08 high 934 aFKBD ra602 ra63 dp ml 4.27 low 935 aFKBDra577 mf dp ml 3.55 low 936 aFKBD ra173 mf dp ml 3.77 low 937 aFKBD ra66mf dp ml 4.44 low 938 aFKBD ra56 mf dp ml 4.43 low 939 aFKBD ra64 mf dpml 4.27 low 940 aFKBD ra171 mf dp ml 4.20 low 941 aFKBD ra63 mf dp ml4.38 low 942 aFKBD ra577 y dp ml 3.23 low 943 aFKBD ra173 y dp ml 3.41low 944 aFKBD ra66 y dp ml 4.06 high 945 aFKBD ra56 y dp ml 4.06 high946 aFKBD ra64 y dp ml 3.93 low 947 aFKBD ra171 y dp ml 3.86 low 948aFKBD ra63 y dp ml 4.01 low 949 aFKBD ra122 mf dp ml 4.13 low 950 aFKBDf ra512 dp ml 4.32 low 951 aFKBD y ra512 dp ml 4.08 low 952 aFKBD mfra512 dp ml 4.44 low 953 aFKBD ra522 ra512 dp ml 4.04 low 954 aFKBDra450 ra512 dp ml 4.12 medium 955 aFKBD ra602 ra348 dp ml 4.09 high 956aFKBD ra602 ra547 dp ml 3.96 high 957 aFKBD ra602 ra381 dp ml 4.01medium 958 aFKBD ra602 ra400 dp ml 4.04 low 959 aFKBD ra602 ra329 dp ml4.03 medium 960 aFKBD ra602 ra325 dp ml 4.09 low 961 aFKBD ra602 ra516dp ml 4.19 low 962 aFKBD ra602 mf dp ra348 4.15 low 963 aFKBD ra602 mfdp ra547 3.99 low 964 aFKBD ra602 mf dp sar 3.70 low 965 aFKBD ra602 mfdp ra147 4.16 high 966 aFKBD ra602 y dp ra348 3.73 low 967 aFKBD ra602 ydp ra547 3.60 low 968 aFKBD ra602 y dp sar 3.17 low 969 aFKBD ra602 y dpra147 3.78 low 970 aFKBD ra602 y dp mi 3.74 medium 971 aFKBD ra512 mf dpml 4.36 low 972 aFKBD ra602 mf dp cha 4.32 low 973 aFKBD ra602 mf dpra84 4.24 low 974 aFKBD ra602 mf dp ra206 3.88 low 975 aFKBD ra602 mf dpra209 4.21 low 976 aFKBD ra602 mf dp ra80 4.21 low 977 aFKBD ra602 mf dpra549 4.57 low 978 aFKBD ra602 mf dp ra189 4.08 medium 979 aFKBD ra602mf dp ra132 3.96 low 980 aFKBD ra602 mf dp mv 4.07 medium 981 aFKBDra602 mf dp ra176 3.52 low 982 aFKBD ra602 mf dp ra301 3.86 low 983aFKBD ra602 mf dp ra81 4.12 low 984 aFKBD ra602 mf dp ra350 4.10 low 985aFKBD ra602 mf dp ra575 4.17 low 986 aFKBD ra602 mf dp ra307 3.74 low987 aFKBD ra602 mf dp ra347 4.20 low 988 aFKBD ra602 mf dp ra554 4.17low 989 aFKBD ra602 mf dp ra546 4.22 low 990 aFKBD ra602 mf dp ra1754.89 low 991 aFKBD ra512 y dp ml 4.06 low 992 aFKBD ra602 y dp cha 4.00low 993 aFKBD ra602 y dp ra84 4.52 low 994 aFKBD ra602 y dp ra206 4.73low 995 aFKBD ra602 y dp ra209 4.12 low 996 aFKBD ra602 y dp ra80 3.91low 997 aFKBD ra602 y dp ra549 4.16 low 998 aFKBD ra602 y dp ra189 3.68low 999 aFKBD ra602 y dp ra132 3.53 low 1000 aFKBD ra602 y dp mv 3.70low 1001 aFKBD ra602 y dp ra176 3.26 low 1002 aFKBD ra602 y dp ra3013.38 low 1003 aFKBD ra602 y dp ra81 3.77 low 1004 aFKBD ra602 y dp ra3503.83 low 1005 aFKBD ra602 y dp ra575 3.85 low 1006 aFKBD ra602 y dpra307 3.25 low 1007 aFKBD ra602 y dp ra347 3.83 low 1008 aFKBD ra602 ydp ra554 4.09 low 1009 aFKBD ra602 y dp ra546 4.74 low 1010 aFKBD ra602y dp ra175 4.79 low 1011 aFKBD ra602 mf ra564 ml 4.97 high 1012 aFKBDra602 mf ra510 ml 4.85 medium 1013 aFKBD ra602 mf ra508 ml 4.49 high1014 aFKBD ra602 mf ra557 ml 4.43 low 1015 aFKBD ra602 mf ra575 ml 4.90low 1016 aFKBD ra602 mf ra81 ml 4.29 low 1017 aFKBD ra602 mf ra554 ml4.79 low 1018 aFKBD ra602 mf ra546 ml 4.84 low 1019 aFKBD ra602 y ra564ml 4.48 medium 1020 aFKBD ra602 y ra510 ml 4.26 high 1021 aFKBD ra602 yra508 ml 4.03 high 1022 aFKBD ra602 y ra557 ml 3.93 low 1023 aFKBD ra602y ra575 ml 4.82 medium 1024 aFKBD ra602 y ra81 ml 5.04 low 1025 aFKBDra602 y ra554 ml 4.31 low 1026 aFKBD ra602 y ra546 ml 4.43 low 1027aFKBD ra602 ra347 dp ml 4.41 high 1028 aFKBD ra602 ra554 dp ml 4.54medium 1029 aFKBD ra602 ra546 dp ml 4.61 low 1030 aFKBD ra602 ra175 dpml 5.45 low 1031 aFKBD ra602 ra307 dp ml 3.86 medium 1032 aFKBD ra602ra522 dp ml 4.07 high 1033 aFKBD ra602 ra206 dp ml 4.12 high 1034 aFKBDra602 ra450 dp ml 4.15 low 1035 aFKBD ra602 ra209 dp ml 4.51 medium 1036aFKBD ra602 ra350 dp ml 4.46 low 1037 aFKBD ra602 ra176 dp ml 3.88 low1038 aFKBD ra602 ra301 dp ml 4.03 low 1039 aFKBD ra602 ra81 dp ml 4.38high 1040 aFKBD ra602 ra549 dp ml 4.94 medium 1041 aFKBD ra602 mv dp ml4.44 high 1042 aFKBD ra602 ra575 dp ml 4.60 low 1043 aFKBD ra602 ra575dp ml 4.47 low 1044 aFKBD ra301 mf dp ml 4.19 low 1045 aFKBD ra347 mf dpml 4.63 low 1046 aFKBD ra554 mf dp ml 4.69 low 1047 aFKBD ra546 mf dp ml4.73 low 1048 aFKBD ra175 mf dp ml 5.81 low 1049 aFKBD ra522 mf dp ml4.18 low 1050 aFKBD ra450 mf dp ml 4.31 high 1051 aFKBD ra549 mf dp ml5.17 low 1052 aFKBD ra176 mf dp ml 3.85 low 1053 aFKBD ra350 mf dp ml4.67 low 1054 aFKBD ra575 mf dp ml 4.15 low 1055 aFKBD ra347 y dp ml4.16 low 1056 aFKBD ra554 y dp ml 4.27 low 1057 aFKBD ra546 y dp ml 4.46low 1058 aFKBD ra175 y dp ml 4.94 low 1059 aFKBD ra522 y dp ml 3.80 low1060 aFKBD ra450 y dp ml 3.91 high 1061 aFKBD ra301 y dp ml 3.80 low1062 aFKBD ra176 y dp ml 3.57 low 1063 aFKBD ra350 y dp ml 4.20 low 1064aFKBD ra575 y dp ml 4.16 low 1065 aFKBD ra513 mf dp ml 4.59 high 1066aFKBD ra602 ra559 dp ml 4.07 high 1067 aFKBD ra602 ra548 dp ml 4.02 high1068 aFKBD ra602 ra536 dp ml 4.07 low 1069 aFKBD ra602 ra576 dp ml 3.63high 1070 aFKBD ra602 dQ dp ml 3.33 low 1071 aFKBD ra602 ra517 dp ml4.06 low 1072 aFKBD ra602 dN dp ml 3.32 low 1073 aFKBD ra602 N dp ml3.35 low 1074 aFKBD ra602 Q dp ml 3.35 medium 1075 aFKBD ra602 ra560 dpml 4.09 high 1076 aFKBD ra602 ra561 dp ml 4.13 low 1077 aFKBD ra602ra534 dp ml 4.15 low 1078 aFKBD ra602 ra382 dp ml 3.98 low 1079 aFKBDra602 ra531 dp ml 4.19 low 1080 aFKBD ra602 ra318 dp ml 4.06 high 1081aFKBD ra602 ra553 dp ml 4.24 medium 1082 aFKBD ra602 ra73 dp ml 4.22 low1083 aFKBD ra602 ra535 dp ml 4.00 low 1084 aFKBD ra602 Aca dp ml 4.42low 1085 aFKBD ra602 ra558 dp ml 4.30 medium 1086 aFKBD ra602 ra529 dpml 3.91 low 1087 aFKBD ra602 ra140 dp ml 3.92 low 1088 aFKBD ra348 mf dpml 4.11 low 1089 aFKBD ra559 mf dp ml 4.25 low 1090 aFKBD ra548 mf dp ml4.14 low 1091 aFKBD ra536 mf dp ml 4.14 low 1092 aFKBD ra576 mf dp ml3.82 low 1093 aFKBD dQ mf dp ml 3.43 low 1094 aFKBD ra517 mf dp ml 4.18low 1095 aFKBD dN mf dp ml 3.44 low 1096 aFKBD N mf dp ml 3.45 low 1097aFKBD Q mf dp ml 3.46 low 1098 aFKBD ra560 mf dp ml 4.24 low 1099 aFKBDra561 mf dp ml 4.24 low 1100 aFKBD ra534 mf dp ml 4.28 low 1101 aFKBDra382 mf dp ml 4.10 low 1102 aFKBD ra531 mf dp ml 4.30 low 1103 aFKBDra318 mf dp ml 4.16 low 1104 aFKBD ra553 mf dp ml 4.33 low 1105 aFKBDra73 mf dp ml 4.32 low 1106 aFKBD ra535 mf dp ml 4.12 low 1107 aFKBD Acamf dp ml 4.53 low 1108 aFKBD ra558 mf dp ml 4.46 low 1109 aFKBD ra529 mfdp ml 4.01 low 1110 aFKBD ra140 mf dp ml 4.04 low 1111 aFKBD ra348 y dpml 3.77 low 1112 aFKBD ra559 y dp ml 3.88 low 1113 aFKBD ra548 y dp ml3.80 low 1114 aFKBD ra536 y dp ml 3.78 low 1115 aFKBD ra576 y dp ml 3.45low 1116 aFKBD dQ y dp ml 3.08 low 1117 aFKBD ra517 y dp ml 3.83 low1118 aFKBD dN y dp ml 3.10 low 1119 aFKBD N y dp ml 3.10 low 1120 aFKBDQ y dp ml 3.12 low 1121 aFKBD ra560 y dp ml 3.91 low 1122 aFKBD ra561 ydp ml 3.88 low 1123 aFKBD ra534 y dp ml 3.94 low 1124 aFKBD ra382 y dpml 3.77 low 1125 aFKBD ra531 y dp ml 3.98 low 1126 aFKBD ra318 y dp ml3.88 low 1127 aFKBD ra553 y dp ml 4.01 low 1128 aFKBD ra73 y dp ml 4.00low 1129 aFKBD ra535 y dp ml 3.77 low 1130 aFKBD Aca y dp ml 4.14 low1131 aFKBD ra558 y dp ml 4.07 low 1132 aFKBD ra529 y dp ml 3.71 low 1133aFKBD ra140 y dp ml 3.70 low 1134 aFKBD ra602 mf ra576 ml 4.00 low 1135aFKBD ra602 mf ra535 ml 4.36 low 1136 aFKBD ra602 mf dN ml 3.66 low 1137aFKBD ra602 mf dQ ml 3.68 high 1138 aFKBD ra602 mf ra536 ml 4.37 low1139 aFKBD ra602 y ra576 ml 3.50 low 1140 aFKBD ra602 y ra535 ml 3.95low 1141 aFKBD ra602 y dN ml 3.18 low 1142 aFKBD ra602 y dQ ml 3.23 low1143 aFKBD ra602 y ra536 ml 3.95 low 1144 aFKBD ra602 mf dp ra559 4.06low 1145 aFKBD ra602 mf dp ra548 4.13 low 1146 aFKBD ra602 mf dp ra5174.14 low 1147 aFKBD ra602 mf dp N 3.46 low 1148 aFKBD ra602 mf dp Q 3.48low 1149 aFKBD ra602 mf dp ra560 4.09 low 1150 aFKBD ra602 mf dp Aca4.53 low 1151 aFKBD ra602 mf dp ra558 4.27 low 1152 aFKBD ra602 y dpra559 3.66 low 1153 aFKBD ra602 y dp ra548 3.69 low 1154 aFKBD ra602 ydp ra517 3.73 low 1155 aFKBD ra602 y dp N 2.42 low 1156 aFKBD ra602 y dpQ 2.57 low 1157 aFKBD ra602 y dp ra560 3.71 low 1158 aFKBD ra602 y dpAca 4.07 low 1159 aFKBD ra602 y dp ra558 3.91 low 1160 aFKBD ra602 mfra545 ml 4.42 high 1161 aFKBD ra602 mf ra102 ml 4.21 medium 1162 aFKBDra602 mf ra351 ml 4.36 low 1163 aFKBD ra602 mf aze ml 3.93 low 1164aFKBD ra602 mf ra529 ml 4.33 low 1165 aFKBD ra602 mf ra140 ml 4.24medium 1166 aFKBD ra602 mf ra538 ml 4.27 low 1167 aFKBD ra602 mf ra603ml 4.15 medium 1168 aFKBD ra602 mf ra528 ml 4.06 medium 1169 aFKBD ra602mf ra532 ml 3.88 low 1170 aFKBD ra602 mf ra539 ml 4.33 high 1171 aFKBDra602 mf ra168 ml 4.09 low 1172 aFKBD ra602 mf ra169 ml 4.19 low 1173aFKBD ra602 mf ra170 ml 3.96 low 1174 aFKBD ra602 mf ra542 ml 4.38 low1175 aFKBD ra602 mf oic ml 4.19 low 1176 aFKBD ra602 mf ra524 ml 3.94low 1177 aFKBD ra602 mf ra165 ml 4.03 medium 1178 aFKBD ra602 mf ra69 ml4.19 low 1179 aFKBD ra602 mf ra573 ml 4.49 low 1180 aFKBD ra602 mf ra574ml 30728.60 low 1181 aFKBD ra602 y ra545 ml 3.96 high 1182 aFKBD ra602 yra102 ml 3.88 low 1183 aFKBD ra602 y ra351 ml 4.01 medium 1184 aFKBDra602 y aze ml 3.48 low 1185 aFKBD ra602 y ra529 ml 3.97 low 1186 aFKBDra602 y ra140 ml 3.89 medium 1187 aFKBD ra602 y ra538 ml 3.89 medium1188 aFKBD ra602 y ra603 ml 3.77 high 1189 aFKBD ra602 y ra528 ml 3.67low 1190 aFKBD ra602 y ra532 ml 3.52 low 1191 aFKBD ra602 y ra539 ml3.98 high 1192 aFKBD ra602 y ra168 ml 3.71 medium 1193 aFKBD ra602 yra169 ml 3.82 high 1194 aFKBD ra602 y ra170 ml 3.52 low 1195 aFKBD ra602y ra542 ml 4.03 high 1196 aFKBD ra602 y oic ml 3.84 low 1197 aFKBD ra602y ra524 ml 3.51 low 1198 aFKBD ra602 y ra165 ml 3.60 medium 1199 aFKBDra602 y ra69 ml 3.82 low 1200 aFKBD ra602 y ra573 ml 4.03 low 1201 aFKBDra602 y ra574 ml 3.87 low 1202 aFKBD ra69 mf dp ml 4.06 low 1203 aFKBDra351 mf dp ml 4.21 low 1204 aFKBD ra102 mf dp ml 4.08 low 1205 aFKBDoic mf dp ml 4.22 low 1206 aFKBD ra542 mf dp ml 4.24 low 1207 aFKBDra574 mf dp ml 4.21 low 1208 aFKBD ra573 mf dp ml 4.30 low 1209 aFKBDra351 y dp ml 3.83 low 1210 aFKBD ra102 y dp ml 3.73 low 1211 aFKBD oicy dp ml 3.78 low 1212 aFKBD ra542 y dp ml 3.81 low 1213 aFKBD ra574 y dpml 3.84 low 1214 aFKBD ra545 y dp ml 3.83 low 1215 aFKBD ra573 y dp ml3.88 low 1216 aFKBD ra602 ra545 dp ml 4.03 low 1217 aFKBD ra602 ra351 dpml 4.89 low 1218 aFKBD ra602 ra69 dp ml 4.10 low 1219 aFKBD ra602 ra102dp ml 3.95 low 1220 aFKBD ra602 y dp mf 3.71 low 1221 aFKBD ra602 mf dpmf 4.07 low 1222 aFKBD ra602 mf dp ra524 3.60 low 1223 aFKBD ra540 mf dpml 4.11 low 1224 aFKBD ra602 y dp ra562 3.72 low 1225 aFKBD ra602 mf dpra562 4.07 low 1226 aFKBD ra602 mf dp y 3.72 low 1227 aFKBD ra602 y dpra542 3.65 low 1228 aFKBD ra602 mf dp ra573 4.15 low 1229 aFKBD ra602 ydp ra573 3.71 low 1230 aFKBD ra602 mf dp ra574 4.03 low 1231 aFKBD ra602rbphe dp ml 3.97 low 1232 aFKBD ra602 ra461 dp ml 3.97 low 1233 aFKBDra602 ra462 dp ml 4.01 low 1234 aFKBD ra602 m dp ml 3.88 high 1235 aFKBDra602 dm dp ml 3.91 low 1236 aFKBD ra602 ra458 dp ml 3.65 medium 1237aFKBD ra602 ra459 dp ml 3.63 medium 1238 aFKBD ra602 ra456 dp ml 3.96high 1239 aFKBD ra602 ra457 dp ml 4.03 low 1240 aFKBD ra602 ra454 dp ml4.00 high 1241 aFKBD ra602 ra321 dp ml 4.01 low 1242 aFKBD ra602 ra452dp ml 3.97 medium 1243 aFKBD ra602 ra306 dp ml 4.02 low 1244 aFKBD ra602ra310 dp ml 4.18 low 1245 aFKBD ra602 ra463 dp ml 4.04 low 1246 aFKBDra602 ra464 dp ml 3.89 low 1247 aFKBD ra602 ra466 dp ml 3.88 low 1248aFKBD ra602 ra467 dp ml 4.01 low 1249 aFKBD ra602 ra468 dp ml 3.94 low1250 aFKBD rbphe mf dp ml 4.02 low 1251 aFKBD ra461 mf dp ml 4.07 low1252 aFKBD ra462 mf dp ml 4.07 low 1253 aFKBD m mf dp ml 4.00 high 1254aFKBD dm mf dp ml 4.00 low 1255 aFKBD ra458 mf dp ml 3.75 low 1256 aFKBDra459 mf dp ml 3.72 low 1257 aFKBD ra456 mf dp ml 4.08 low 1258 aFKBDra457 mf dp ml 4.09 low 1259 aFKBD ra454 mf dp ml 4.10 low 1260 aFKBDra321 mf dp ml 4.07 low 1261 aFKBD ra452 mf dp ml 4.08 low 1262 aFKBDra306 mf dp ml 4.07 low 1263 aFKBD ra453 mf dp ml 4.16 low 1264 aFKBDra310 mf dp ml 4.29 low 1265 aFKBD ra463 mf dp ml 4.21 low 1266 aFKBDra464 mf dp ml 4.01 low 1267 aFKBD ra466 mf dp ml 4.01 low 1268 aFKBDra467 mf dp ml 4.13 low 1269 aFKBD ra468 mf dp ml 4.10 low 1270 aFKBDrbphe y dp ml 3.69 low 1271 aFKBD ra461 y dp ml 3.71 low 1272 aFKBDra462 y dp ml 3.73 low 1273 aFKBD m y dp ml 3.64 high 1274 aFKBD dm y dpml 3.64 low 1275 aFKBD ra458 y dp ml 3.43 low 1276 aFKBD ra459 y dp ml3.42 low 1277 aFKBD ra456 y dp ml 3.77 low 1278 aFKBD ra457 y dp ml 3.77low 1279 aFKBD ra454 y dp ml 3.76 low 1280 aFKBD ra321 y dp ml 3.75 low1281 aFKBD ra452 y dp ml 3.77 low 1282 aFKBD ra306 y dp ml 3.77 low 1283aFKBD ra453 y dp ml 3.86 low 1284 aFKBD ra310 y dp ml 3.91 low 1285aFKBD ra463 y dp ml 3.85 low 1286 aFKBD ra464 y dp ml 3.65 low 1287aFKBD ra466 y dp ml 3.69 low 1288 aFKBD ra467 y dp ml 3.83 low 1289aFKBD ra468 y dp ml 3.80 low 1290 aFKBD phg mf dp rbphe 3.86 low 1291aFKBD phg mf dp ra461 3.95 low 1292 aFKBD ra602 mf dp ra462 3.97 low1293 aFKBD ra602 mf dp m 3.96 low 1294 aFKBD ra602 mf dp ra458 3.73 low1295 aFKBD ra602 mf dp ra456 4.12 low 1296 aFKBD ra602 mf dp ra454 4.07low 1297 aFKBD ra602 mf dp ra452 4.06 low 1298 aFKBD ra602 mf dp ra4534.00 high 1299 aFKBD ra602 mf dp ra310 4.32 low 1300 aFKBD ra602 mf dpra463 3.98 low 1301 aFKBD ra602 y dp rbphe 3.54 low 1302 aFKBD ra602 ydp ra461 3.56 low 1303 aFKBD ra602 y dp ra462 3.55 low 1304 aFKBD ra602y dp m 3.51 low 1305 aFKBD ra602 y dp ra458 3.21 low 1306 aFKBD ra602 ydp ra456 3.64 low 1307 aFKBD ra602 y dp ra454 3.64 low 1308 aFKBD ra602y dp ra452 3.65 low 1309 aFKBD ra602 y dp ra453 3.66 low 1310 aFKBDra602 y dp ra310 3.86 low 1311 aFKBD ra602 y dp ra463 3.66 low 1312aFKBD ra602 mf dm ml 4.23 high 1313 aFKBD ra602 mf ra459 ml 3.92 high1314 aFKBD ra602 mf ra457 ml 4.27 low 1315 aFKBD ra602 mf ra321 ml 4.26low 1316 aFKBD ra602 mf ra306 ml 4.26 medium 1317 aFKBD ra602 mf ra463ml 4.25 low 1318 aFKBD ra602 y dm ml 3.79 low 1319 aFKBD ra602 y ra459ml 3.50 medium 1320 aFKBD ra602 y ra457 ml 3.90 low 1321 aFKBD ra602 yra321 ml 3.90 low 1322 aFKBD ra602 y ra306 ml 3.89 low 1323 aFKBD ra602y ra463 ml 3.91 low 1324 aFKBD ra602 ra110 dp ml 4.30 low 1325 aFKBDra602 ra115 dp ml 4.02 medium 1326 aFKBD ra602 ra117 dp ml 4.08 high1327 aFKBD ra602 ra116 dp ml 4.08 medium 1328 aFKBD ra602 ra113 dp ml3.90 medium 1329 aFKBD ra602 ra114 dp ml 3.87 high 1330 aFKBD ra602ra112 dp ml 3.85 high 1331 aFKBD ra602 ra111 dp ml 3.56 low 1332 aFKBDra602 mf dp mi 4.13 medium 1333 aFKBD ra602 ra148 dp ml 4.13 medium 1334aFKBD ra602 napA dp ml 4.10 medium 1335 aFKBD ra602 tic dp ml 3.95 low1336 aFKBD ra602 ra136 dp ml 3.67 low 1337 aFKBD ra602 ra105 dp ml 3.67low 1338 aFKBD ra602 ra137 dp ml 4.14 medium 1339 aFKBD ra602 ra101 dpml 3.89 low 1340 aFKBD ra602 ra540 dp ml 4.04 low 1341 aFKBD ra602 ra86dp ml 4.04 low 1342 aFKBD ra602 ra204 dp ml 4.04 low 1343 aFKBD ra602ra134 dp ml 4.04 high 1344 aFKBD ra602 ra135 dp ml 4.20 low 1345 aFKBDra602 ra525 dp ml 4.12 low 1346 aFKBD ra602 ra122 dp ml 4.00 medium 1347aFKBD ra122 ra122 dp ml 4.10 low 1348 aFKBD ra122 y dp ml 3.76 low 1349aFKBD ra110 mf dp ml 4.41 low 1350 aFKBD ra115 mf dp ml 4.14 low 1351aFKBD ra117 mf dp ml 4.20 low 1352 aFKBD ra116 mf dp ml 4.18 low 1353aFKBD ra113 mf dp ml 4.00 low 1354 aFKBD ra114 mf dp ml 4.00 low 1355aFKBD ra112 mf dp ml 3.96 low 1356 aFKBD ra111 mf dp ml 3.72 low 1357aFKBD ra109 mf dp ml 3.60 low 1358 aFKBD ra108 mf dp ml 3.55 low 1359aFKBD ra148 mf dp ml 4.24 low 1360 aFKBD napA mf dp ml 4.24 low 1361aFKBD ra602 mf dp ml 4.05 high 1362 aFKBD ra136 mf dp ml 3.79 low 1363aFKBD ra105 mf dp ml 3.81 low 1364 aFKBD ra137 mf dp ml 4.27 low 1365aFKBD ra101 mf dp ml 4.08 low 1366 aFKBD ra86 mf dp ml 4.39 low 1367aFKBD ra134 mf dp ml 4.11 low 1368 aFKBD ra135 mf dp ml 4.26 low 1369aFKBD ra525 mf dp ml 4.17 low 1370 aFKBD ra110 y dp ml 4.05 low 1371aFKBD ra115 y dp ml 3.79 low 1372 aFKBD ra117 y dp ml 3.83 low 1373aFKBD ra116 y dp ml 3.84 medium 1374 aFKBD ra113 y dp ml 3.68 low 1375aFKBD ra114 y dp ml 3.66 low 1376 aFKBD ra112 y dp ml 3.64 low 1377aFKBD ra111 y dp ml 3.40 low 1378 aFKBD ra109 y dp ml 3.26 low 1379aFKBD ra108 y dp ml 3.20 low 1380 aFKBD ra148 y dp ml 3.87 low 1381aFKBD napA y dp ml 3.88 low 1382 aFKBD ra136 y dp ml 3.50 low 1383 aFKBDra105 y dp ml 3.43 low 1384 aFKBD ra540 y dp ml 3.77 low 1385 aFKBD ra86y dp ml 3.74 low 1386 aFKBD ra204 y dp ml 3.70 low 1387 aFKBD ra134 y dpml 3.76 low 1388 aFKBD ra135 y dp ml 3.94 low 1389 aFKBD ra525 y dp ml3.86 low 1390 aFKBD ra602 mf ra540 ml 4.23 medium 1391 aFKBD ra602 yra540 ml 3.75 low 1392 aFKBD ra602 y ra86 ml 4.16 low 1393 aFKBD ra602mf tic ml 4.15 low 1394 aFKBD ra602 y tic ml 3.75 low 1395 aFKBD ra602mf ra105 ml 3.95 high 1396 aFKBD ra602 y ra105 ml 3.63 high 1397 aFKBDra602 mf ra136 ml 3.87 low 1398 aFKBD ra602 y ra136 ml 3.54 low 1399aFKBD ra602 ra513 dp ml 5.67 high 1400 aFKBD ra602 ra120 dp ml 4.88 low1401 aFKBD ra602 ra92 dp ml 5.10 low 1402 aFKBD ra602 ra107 dp ml 5.14high 1403 aFKBD ra602 ra93 dp ml 5.14 medium 1404 aFKBD ra602 ra95 dp ml5.28 low 1405 aFKBD ra602 ra96 dp ml 5.23 medium 1406 aFKBD ra602 ra87dp ml 4.91 medium 1407 aFKBD ra602 ra104 dp ml 4.91 high 1408 aFKBDra602 ra123 dp ml 4.90 high 1409 aFKBD ra602 ra89 dp ml 3.55 high 1410aFKBD ra602 ra90 dp ml 3.67 medium 1411 aFKBD ra602 ra91 dp ml 4.02medium 1412 aFKBD ra602 ra97 dp ml 5.25 low 1413 aFKBD ra602 ra94 dp ml5.29 low 1414 aFKBD ra602 ra353 dp ml 5.43 medium 1415 aFKBD ra602 ra88dp ml 4.80 high 1416 aFKBD ra602 ra185 dp ml 4.92 high 1417 aFKBD ra602ra124 dp ml 4.81 high 1418 aFKBD ra602 ra526 dp ml 5.07 high 1419 aFKBDra602 ra121 dp ml 4.86 high 1420 aFKBD ra602 ra339 dp ml 4.91 high 1421aFKBD ra602 ra106 dp ml 4.59 high 1422 aFKBD ra602 my dp ml 4.58 high1423 aFKBD ra602 ra133 dp ml 4.40 high 1424 aFKBD ra602 mf dp ra83 4.16low 1425 aFKBD ra92 mf dp ml 5.26 low 1426 aFKBD ra107 mf dp ml 5.27 low1427 aFKBD ra93 mf dp ml 5.32 low 1428 aFKBD ra95 mf dp ml 5.43 low 1429aFKBD ra96 mf dp ml 5.44 low 1430 aFKBD Ra87 mf dp ml 5.15 low 1431aFKBD ra602 ra108 dp ml 3.46 high 1432 aFKBD ra123 mf dp ml 5.15 low1433 aFKBD ra89 mf dp ml 3.58 low 1434 aFKBD ra90 mf dp ml 3.66 low 1435aFKBD ra97 mf dp ml 5.45 low 1436 aFKBD ra94 mf dp ml 5.38 low 1437aFKBD ra353 mf dp ml 5.60 low 1438 aFKBD ra88 mf dp ml 4.94 low 1439aFKBD ra185 mf dp ml 5.06 low 1440 aFKBD ra124 mf dp ml 5.00 low 1441aFKBD ra526 mf dp ml 5.21 low 1442 aFKBD ra121 mf dp ml 5.02 low 1443aFKBD ra119 mf dp ml 5.06 low 1444 aFKBD ra339 mf dp ml 5.05 low 1445aFKBD ra106 mf dp ml 4.79 low 1446 aFKBD my mf dp ml 4.63 low 1447 aFKBDra133 mf dp ml 4.55 low 1448 aFKBD ra513 y dp ml 4.10 high 1449 aFKBDra120 y dp ml 4.51 high 1450 aFKBD ra92 y dp ml 4.72 low 1451 aFKBDra107 y dp ml 4.79 low 1452 aFKBD ra93 y dp ml 4.80 low 1453 aFKBD ra95y dp ml 4.91 low 1454 aFKBD ra96 y dp ml 4.92 low 1455 aFKBD Ra87 y dpml 4.58 low 1456 aFKBD ra104 y dp ml 4.59 low 1457 aFKBD ra123 y dp ml4.58 low 1458 aFKBD ra89 y dp ml 3.06 low 1459 aFKBD ra90 y dp ml 3.24low 1460 aFKBD ra91 y dp ml 3.20 low 1461 aFKBD ra97 y dp ml 4.77 low1462 aFKBD ra94 y dp ml 4.76 low 1463 aFKBD ra353 y dp ml 5.14 low 1464aFKBD ra88 y dp ml 4.42 low 1465 aFKBD ra185 y dp ml 4.49 low 1466 aFKBDra124 y dp ml 4.44 low 1467 aFKBD ra526 y dp ml 4.75 low 1468 aFKBDra121 y dp ml 4.47 low 1469 aFKBD ra119 y dp ml 4.50 low 1470 aFKBDra339 y dp ml 4.49 medium 1471 aFKBD ra106 y dp ml 4.25 low 1472 aFKBDmy y dp ml 4.16 low 1473 aFKBD ra133 y dp ml 4.03 low 1474 raa26 ra602mf dp ml 6.14 high 1475 raa26 ra602 y dp ml 5.89 high 1476 raa21 ra602 ydp ml 3.91 high 1477 raa21 ra602 mf dp ml 5.99 high 1478 raa7 ra602 mfdp ml 5.15 medium 1479 raa7 ra602 y dp ml 4.08 low 1480 raa6 ra602 mf dpml 6.33 high 1481 raa6 ra602 y dp ml 6.38 high 1482 raal ra602 mf dp ml4.47 high 1483 raal ra602 y dp ml 4.47 low 1484 raa25 ra602 mf dp ml5.90 high 1485 raal4 ra602 mf dp ml 7.44 low 1486 raal4 ra602 y dp ml6.60 low 1487 raal6 ra602 mf dp ml 7.30 low 1488 raal6 ra602 y dp ml6.52 low 1489 raal2 ra602 mf dp ml 6.10 high 1490 raal2 ra602 y dp ml5.51 high 1491 raa3 ra602 mf dp ml 5.88 low 1492 raa3 ra602 y dp ml 5.28low 1493 aFKBD ra602 ra109 dp ml 3.50 high 1494 raal3 ra602 y dp ml 6.65low 1495 raall ra602 mf dp ml 6.29 high 1496 raall ra602 y dp ml 4.66high 1497 raal5 ra602 mf dp ml 5.17 low 1498 raal5 ra602 y dp ml 4.70low 1499 raa4 ra602 mf dp ml 4.69 low 1500 raa4 ra602 y dp ml 5.39 low1501 raa31 ra602 mf dp ml 5.00 medium 1502 raa29 ra602 mf dp ml 5.22high 1503 raa29 ra602 y dp ml 4.59 medium 1504 raa32 ra602 mf dp ml 5.66medium 1505 raa8 ra602 mf dp ml 4.71 high 1506 raal0 ra602 mf dp ml 4.91high 1507 raa8 ra602 y dp ml 5.15 medium 1508 raal0 ra602 y dp ml 4.19low 1509 raa2 ra602 mf dp ml 4.76 medium 1510 raa2 ra602 y dp ml 5.91low 1511 raa5 ra602 mf dp ml 5.26 low 1512 raa5 ra602 y dp ml 4.60 low1513 aFKBD ra602 ra119 dp ml 4.91 high 1514 aFKBD ra602 ra520 dp ml 4.31high 1515 aFKBD ra602 ra569 dp ml 4.10 medium 1516 aFKBD ra602 ra570 dpml 4.01 low 1517 aFKBD ra602 ra571 dp ml 4.01 low 1518 aFKBD ra602 ra572dp ml 3.95 low 1519 aFKBD ra602 ra399 dp ml 4.71 low 1520 aFKBD ra602ra515 dp ml 5.34 low 1521 aFKBD ra602 ra398 dp ml 6.89 low 1522 aFKBDra602 y dp ml 3.65 high 1523 raa9 ra602 mf dp ml 4.02 low 1524 aFKBDra132 mf dp ml 5.76 low 1525 aFKBD ra127 mf dp ml 5.46 high 1526 aFKBDra126 mf dp ml 5.39 low 1527 aFKBD ra189 mf dp ml 5.91 medium 1528 aFKBDra84 mf dp ml 5.19 high 1529 aFKBD ra83 mf dp ml 5.92 medium 1530 aFKBDra130 mf dp ml 6.01 low 1531 aFKBD ra600 mf dp ml 5.88 high 1532 aFKBDra565 mf dp ml 5.97 low 1533 aFKBD ra602 y dp ra83 4.44 low 1534 aFKBDtic mf dp ml 4.10 low 1535 aFKBD ra147 mf dp ml 6.18 low 1536 aFKBDra563 mf dp ml 6.14 low 1537 aFKBD ra602 mf dp ml 5.83 low 1538 raal3ra602 mf dp ml 7.41 low 1539 raal9 ra602 mf dp ml 5.46 low 1540 raal9ra602 y dp ml 4.75 low 1541 raa20 ra602 mf dp ml 6.31 low 1542 raa22ra602 ra471 dp ml 3.31 medium 1543 aFKBD ra602 ra472 dp ml 3.70 high1544 aFKBD ra602 ra471 dp ml 5.26 high 1545 aFKBD ra602 mf ra473 ml 6.57low 1546 aFKBD ra602 y ra473 ml 3.07 low 1547 aFKBD ra602 ra512 ra105 ml6.45 high 1548 aFKBD ra513 ra512 ra105 ml 6.06 medium 1549 aFKBD ra513mf ra105 ml 5.84 medium 1550 raa20 ra602 y dp ml 5.78 low 1551 aFKBDra513 ra512 dp ml 6.23 low 1552 aFKBD ra602 ra511 dp ml 6.59 medium 1553aFKBD ra513 ra520 dp ml 5.13 medium 1554 aFKBD ra513 ra520 ra105 ml 4.13high 1555 raa18 ra602 mf dp ml 4.39 high 1556 rae27 ra602 mf dp ml 5.02low 1557 raa17 ra602 mf dp ml 4.37 high 1558 afkbd phg ra500 dp ml 3.81high 1559 afkbd phg ra501 dp ml 3.86 medium 1560 afkbd phg ra502 dp ml3.83 low 1561 afkbd phg ra503 dp ml 3.19 low 1562 afkbd phg ra504 dp ml3.22 low 1563 rae21 ra147 napA ra562 g 6.94 high 1564 rae29 ra147 napAra562 g 6.67 high 1565 rae26 ra147 napA ra562 g low 1566 rae1 my df sardf medium 1567 rae10 my df sar df medium 1568 rae11 my df sar df low1569 rae12 my df sar df low 1570 rae13 my df sar df medium 1571 rae14 mydf sar df low 1572 rae16 my df sar df low 1573 rae16a my df sar df low1574 rae17 my df sar df low 1575 rae18 my df sar df low 1576 rae19 my dfsar df medium 1577 rae2 my df sar df medium 1578 rae20 my df sar df low1579 rae21 my df sar df medium 1580 rae26 my df sar df low 1581 rae3 mydf sar df medium 1582 rae4 my df sar df low 1583 rae5 my df sar df low1584 rae9 my df sar df low 1585 afkbd phg ra655 dp ml 3.72 High 1586afkbd phg ra656 dp ml 3.74 Med 1587 afkbd phg ra626 dp ml 3.15 Low 1588afkbd phg ra592 dp ml 3.44 High 1589 afkbd phg ra618 dp ml 3.10 Low 1590afkbd phg ra655 dp ml 3.72 High 1591 afkbd phg ra656 dp ml 3.74 Med 1592afkbd phg ra626 dp ml 3.15 Low 1593 afkbd phg ra592 dp ml 3.44 High 1594afkbd phg ra618 dp ml 3.10 Low 1595 afkbd phg ra620 dp ml 3.92 Low 1596afkbd phg ra623 dp ml 3.96 Low 1597 afkbd ml df mi g 6.48 High 1598aFKBD Ra602 Ra503 dp ml 5.09 high 1599 aFKBD mf dp ml 5.83 low 1600aFKBD Ra602 mf ml 4.01 low 1601 aFKBD Ra602 y ml 3.53 low 1602 aFKBD ydp ml 3.57 low 1603 aFKBD Ra195 dp ml 4.02 low 1604 aFKBD mf dp ml 4.49low

TABLE 8 Rapafucin compound 1605 to compound 1627. Com- Uptake, poundHill- IC50 No. RA1 RA2 RA3 RA4 RA5 slope (nM) 1605 aFKBD Gly dmPhe PromVal −0.9753 27.95 1606 aFKBD Ala dmPhe Pro mVal −1.164 23.73 1607 aFKBDNva dmPhe Pro mVal −1.112 18 1608 aFKBD Leu dmPhe Pro mVal −1.105 54.141609 aFKBD Phe dmPhe Pro mVal −1.191 54.99 1610 aFKBD Phg dmPhe Pro mVal−0.8952 16.51 1611 eFKBD Gly dmPhe Pro mVal −1.024 48.88 1612 eFKBD AladmPhe Pro mVal −1.125 33.54 1613 eFKBD HoSMe dmPhe Pro mVal −0.861459.46 1614 aFKBD Ala dmPhe Pro mlle −0.6276 34.4 1615 aFKBD Nva dmPhePro mlle −0.87 12.19 1616 aFKBD Phg dmPhe Pro mlle −0.9138 100.1 1617eFKBD Ala dmPhe Pro mlle −1.212 34.15 1618 eFKBD Nva dmPhe Pro mlle−1.195 173.1 1619 eFKBD Ala dmPhe Pro mAla −1.134 66.71 1620 eFKBD GlydmPhe Pro mNIe −1.007 13.91 1621 eFKBD Ala dmPhe Pro mNIe −1.017 9.761622 eFKBD Gly dmPhe Pro mLeu −1.494 28.54 1623 eFKBD Ala dmPhe Pro mLeu−0.741 10.53 1624 aFKBD Ala dmPhe Pro mLeu −0.3876 31.45 1625 eFKBD GlydmPhe Pro mNva −1.363 42.27 1626 eFKBD Gly dmPhe Pro dmAla −1.314 154.91627 eFKBD Gly dmPhe Pro Ach −1.236 261.9

In treatment, the dose of agent optionally ranges from about 0.0001mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.15mg/kg to about 3 mg/kg, 0.5 mg/kg to about 2 mg/kg and about 1 mg/kg toabout 2 mg/kg of the subject's body weight. In other embodiments thedose ranges from about 100 mg/kg to about 5 g/kg, about 500 mg/kg toabout 2 mg/kg and about 750 mg/kg to about 1.5 g/kg of the subject'sbody weight. For example, depending on the type and severity of thedisease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of agent is acandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage is in the range from about 1 μg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be useful.Unit doses can be in the range, for instance of about 5 mg to 500 mg,such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg and 300 mg. The progressof therapy is monitored by conventional techniques and assays.

In some embodiments, an agent is administered to a human patient at aneffective amount (or dose) of less than about 1 μg/kg, for instance,about 0.35 to about 0.75 μg/kg or about 0.40 to about 0.60 μg/kg. Insome embodiments, the dose of an agent is about 0.35 μg/kg, or about0.40 μg/kg, or about 0.45 μg/kg, or about 0.50 μg/kg, or about 0.55μg/kg, or about 0.60 μg/kg, or about 0.65 μg/kg, or about 0.70 μg/kg, orabout 0.75 μg/kg, or about 0.80 μg/kg, or about 0.85 μg/kg, or about0.90 μg/kg, or about 0.95 μg/kg or about 1 μg/kg. In variousembodiments, the absolute dose of an agent is about 2 μg/subject toabout 45 μg/subject, or about 5 to about 40, or about 10 to about 30, orabout 15 to about 25 μg/subject. In some embodiments, the absolute doseof an agent is about 20 μg, or about 30 μg, or about 40 μg.

In various embodiments, the dose of an agent may be determined by thehuman patient's body weight. For example, an absolute dose of an agentof about 2 μg for a pediatric human patient of about 0 to about 5 kg(e.g., about 0, or about 1, or about 2, or about 3, or about 4, or about5 kg); or about 3 μg for a pediatric human patient of about 6 to about 8kg (e.g., about 6, or about 7, or about 8 kg), or about 5 μg for apediatric human patient of about 9 to about 13 kg (e.g., 9, or about 10,or about 11, or about 12, or about 13 kg); or about 8 μg for a pediatrichuman patient of about 14 to about 20 kg (e.g., about 14, or about 16,or about 18, or about 20 kg), or about 12 μg for a pediatric humanpatient of about 21 to about 30 kg (e.g., about 21, or about 23, orabout 25, or about 27, or about 30 kg), or about 13 μg for a pediatrichuman patient of about 31 to about 33 kg (e.g., about 31, or about 32,or about 33 kg), or about 20 μg for an adult human patient of about 34to about 50 kg (e.g., about 34, or about 36, or about 38, or about 40,or about 42, or about 44, or about 46, or about 48, or about 50 kg), orabout 30 μg for an adult human patient of about 51 to about 75 kg (e.g.,about 51, or about 55, or about 60, or about 65, or about 70, or about75 kg), or about 45 μg for an adult human patient of greater than about114 kg (e.g., about 114, or about 120, or about 130, or about 140, orabout 150 kg).

In certain embodiments, an agent in accordance with the methods providedherein is administered subcutaneously (s.c.), intraveneously (i.v.),intramuscularly (i.m.), intranasally or topically. Administration of anagent described herein can, independently, be one to four times daily orone to four times per month or one to six times per year or once everytwo, three, four or five years. Administration can be for the durationof one day or one month, two months, three months, six months, one year,two years, three years, and may even be for the life of the humanpatient. The dosage may be administered as a single dose or divided intomultiple doses. In some embodiments, an agent is administered about 1 toabout 3 times (e.g., 1, or 2 or 3 times).

The following example is provided to further illustrate the advantagesand features of the present disclosure, but it is not intended to limitthe scope of the disclosure. While this example is typical of those thatmight be used, other procedures, methodologies, or techniques known tothose skilled in the art may alternatively be used.

EXAMPLES

General experimental for synthesis. Syntheti reagents. Piperidine,N,N-diisopropylethylamine (DIPEA) were purchased from Alfa Aesar.Anhydrous pyridine was purchased from Acros. Solid support resin with2-chlorotrityl chloride (Cat#: 03498) was purchased from Chem-Impex.HATU was purchased from ChemImpex. Fmoc protected amino acid buildingblocks were purchased from ChemImpex, Novabiochem or GL Biochem.Dichloromethane (DCM or CH₂Cl₂), methanol (MeOH), hexanes, ethyl acetate(EtOAc), 1,2-dichloroethane (DCE, anhydrous), N,N′-dimethylformamide(DMF, anhydrous), Hoveyda-Grubbs catalyst 2nd generation and all theother chemical reagents were purchased from Sigma-Aldrich.

Instruments for synthesis and purification. NMR spectra were recordedwith Burker-400 and -500. High performance liquid chromatographicanalyses were performed with Agilent LC-MS system (Agilent 1260 series,mass detector 6120 quadrupole). Orbital shaking for solid-phasereactions was performed on a Mettler-Toledo Bohdan MiniBlock system for96 tubes (30-200 mg resin in SiliCycle tubes) or a VWR Mini Shaker(0.2-2 g resin in a plastic syringe with a fritted disc). Reagents wereadded with an adjustable Rainin 8-channel pipette for the MiniBlocksystem. Microwave reactions were performed with a Biotage Initiator Plusor Multiwave Pro with silicon carbide 24-well blocks from Anton Parr.Compound purification at 0.05-50 g scale was performed with TeledyneIsco CombiFlash Rf 200 or Biotage Isolera One systems followed by aHeidolph rotary evaporator. Purification at 1-50 mg scale was performedwith Agilent HPLC system. Mixture of Rapafucins in the 45,000-compoundlibrary are purified in a high-throughput manner by SPE cartridges(Biotage, 460-0200-C, ISOLUTE, SI 2 g/6 mL) on vacuum manifold(Sigma-Aldrich, Visiprep™ SPE Vacuum Manifold, Disposable Liner,12-port) followed by overnight drying with a custom-designed box (50cm×50 cm×15 cm) that allows air flowing rapidly inside to remove thesolvent. The high-throughput weighing of the compounds in the librarywas done by a Mettler-Toledo analytical balance that linked (SartoriousEntris line with RS232 port) to a computer with custom-coded electronicspreadsheet.

FKBD Example 14-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoicacid (aFKBD)

2-allyl 1-(tert-butyl) (S)-piperidine-1,2-dicarboxylate (2). To asolution of N-Boc homoproline 1 (6.30 g) in DMF (40 mL), Cs₂CO₃ (2.90 g)was added. The resulting suspension was stirred at RT for 5 min beforethe addition of allyl bromide (6.3 g). After stirring at RT for 2 h, thesuspension was filtered through a pad of celite, rinsed with EtOAc (50mL), and washed with HCl (1M, 50 mL×3). The organic layer was dried overNa₂SO₄ and co-evaporated with toluene (30 mL×2). Crude product (8.10 g)was collected as a yellow oil and was pure enough for the next stepwithout further purification. The crude product (8.10 g) and TFA (4.3 g)were mixed well in dichloromethane (20 mL) and stirred at RT for 0.5 h.2-allyl 1-(tert-butyl) (S)-piperidine-1,2-dicarboxylate 2 (3.00 g) wascollected as a yellow oil and was pure enough for the next step withoutfurther purification.

allyl(S)-1-(4-hydroxy-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(3). Compound 2 (3.0 g), dihydro-4,4-dimethyl-2,3-furandione (2.1 g) andDMAP (20 mg) were dissolved in toluene (20 mL) and the reaction wasrefluxed with an oil bath (120° C.) for 14 h. After the solvent wasremoved, the residue was purified by column chromatography (80-200 mesh)with EtOAc/hexane (1/3). 3 (3.50 g) was collected as a yellow oil. ¹HNMR (500 MHz, CDCl₃) δ 6.04-5.80 (m, 1H), 5.36 (d, J=17 Hz, 1H),5.31-5.25 (m, 2H), 4.68 (s, 2H), 3.76-3.56 (m, 2H), 3.50 (d, J=13 Hz,1H), 3.40 (s, 1H), 3.20 (t, J=13 Hz, 1H), 2.37 (d, J=13 Hz, 1H),1.84-1.61 (m, 3H), 1.61-1.34 (m, 2H), 1.24 (s, 6H). ¹³C NMR (126 MHz,CDCl₃) δ 205.9, 170.1, 168.1, 131.4, 119.2, 69.3, 66.3, 51.6, 49.5,44.2, 26.3, 24.8, 21.3, 21.2, 21.0.

allyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(4) Acryloyl chloride (0.78 g) in dry CH₂Cl₂ (20 mL) was added dropwiseto a mixture of compound 3 (3.50 g) and N,N-diisopropylethylamine (2.0mL) in 50 mL CH₂Cl₂ with ice-batch over 30 min. After addition, thereaction was allowed to stir at RT for 30 min before quenched withsaturated NaHCO₃ solution (20 mL). The organic phase was washed withwater, dried over Na₂SO₄, concentrated and purified by column (EtOAc:Hexans=1:5) to afford product 4 (2.21 g) as a yellow oil. ¹H NMR (500MHz, CDCl₃) δ 6.39 (dd, J=17, 1.5 Hz, 1H), 6.08 (dd, J=17,11 Hz, 1H),5.91 (ddt, J=17, 11, 6 Hz, 1H), 5.84 (dd, J=11, 1.5 Hz, 1H), 5.35 (ddd,J=17, 2.5, 1.5 Hz, 1H), 5.28-5.25 (m, 1H), 5.26 (ddd, J=11, 2.5, 1.5 Hz,1H), 4.66 (ddd, J=6, 4, 2.5 Hz, 2H), 4.37 (d, J=11 Hz, 1H), 4.27 (d,J=11 Hz, 1H), 3.52 (dd, J=13, 1.5 Hz, 1H), 3.23 (td, J=13, 3 Hz, 1H),2.34 (d, J=14 Hz, 1H), 1.84-1.76 (m, 1H), 1.76-1.67 (m, 1H), 1.67-1.60(m, 1H), 1.59-1.47 (m, 1H), 1.47-1.38 (m, 1H), 1.36 (s, 3H), 1.35 (s,3H). ¹³C NMR (126 MHz, CDCl₃) δ 204.8, 169.8, 166.7, 165.5, 131.5,131.2, 128.0, 118.9, 69.5, 69.3, 66.0, 51.3, 46.7, 43.9, 26.4, 24.9,22.2, 21.5, 21.1. HRMS for [M+H]+ C18H25NO6, calculated: 352.1760,observed: 352.1753.

(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylicacid (5). compound 4 (4.2 g), Pd(PPh3)4 (230 mg), N-methylaniline (2.5mL) were dissolved in THF (40 mL) and stirred at RT for 6 h. Thereaction mixture was then diluted with EtOAc (80 mL) and washed with HCl(1M, 50 mL×3). The organic phase was separated, dried over Na₂SO₄,filtered and concentrated. The crude product was purified using columnchromatography (200-400 mesh), where the byproduct can be eluted with 2%MeOH in dichloromethane, followed by the desired product with 3% MeOHand 0.1% AcOH in dichloromethane. 5 (2.55 g) was collected as a whitesolid (66%). ¹H NMR (500 MHz, CDCl₃) δ 9.96 (s, 1H), 6.39 (d, J=17 Hz,1H), 6.08 (dd, J=17,10Hz, 1H), 5.85 (d, J=10 Hz, 1H), 5.30 (s, 1H),4.55-4.30 (m, 1H), 4.32 (d, J=6 Hz, 2H), 3.53 (d, J=12 Hz, 1H), 3.24 (t,J=12 Hz, 1H), 2.35 (d, J=13 Hz, 1H), 1.91-1.60 (m, 2H), 1.60-1.42 (m,2H), 1.36 (s, 3H), 1.34 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 204.7,175.3, 166.8, 165.7, 131.4, 127.8, 69.6, 69.5, 51.2, 46.7, 44.0, 26.2,24.9, 22.1, 21.8, 21.1. HRMS for [M+H]+ C15H21NO6, calculated: 312.1447,observed: 312.1444.

(E)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6). To asolution of 3,4-dimethoxybenzaldehyde (5.10 g) and 3-amino acetophenone(4.15 g) mixture in EtOH (20 mL, 95%), NaOH (0.2 g in 2 mL water) wasadded. The reaction mixture was stirred at RT for 6 h and a slurry ofyellow precipitate was formed. The reaction mixture was then dilutedwith EtOAc (40 mL) and washed with water (30 mL×3). Upon concentrated,the crude product 6 (9.0 g) is pure enough for the next step.

1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propan-1-one (7). To a solutionof α,β-unsaturated ketone 6 (crude, 9.0 g) in MeOH (20 mL), Pd/C (10%,1.61 g) was added. The reaction vessel was flushed with hydrogen gasrepetitively by using a balloon of hydrogen and high vacuum. Thereaction mixture was stirred at RT for 1 h before filtered through a padof celite. Longer reaction time would render the reaction to generateundesired byproducts. The filtrate was concentrated and subject tocolumn chromatography (50 g silica gel) and eluted withEtOAc/CH₂Cl₂/hexane (1/3/3 to 1/1/1). 7 (2.48 g) was collected as ayellow oil. ¹H NMR (500 MHz, CDCl₃) δ 7.36-7.16 (m, 3H, ar), 6.92-6.71(m, 4H, ar), 3.86 (s, 3H, OCH₃), 3.85 (s, 3H, OCH3), 3.81 (s, 2H, NH2),3.23 (t, J=7.5 Hz, 2H, COCH2), 2.99 (t, J=7.4 Hz, 2H, ArCH2). ¹³C NMR(126 MHz, CDCl₃) δ 199.66 (C═O), 148.90 (ar), 147.38 (ar), 146.82 (ar),138.03 (ar), 134.03 (ar), 129.49 (ar), 120.19 (ar), 119.61 (ar), 118.44(ar), 113.91 (ar), 111.87 (ar), 111.35 (ar), 55.98 (OCH₃), 55.87 (OCH3),40.80 (COCH2), 29.91 (ArCH2). HRMS for [M+H]+ C17H19NO₃, calculated:286.1443, observed: 286.1436.

4-((3-(3-(3,4-dimethoxyphenyl)propanoyl)phenyl)amino)-4-oxobutanoic acid(9). Aniline 7 (3.50 g), succinic anhydride (1.0 g) and DMAP (61 mg)were mixed in dichloromethane (30 mL). After stirring at RT for 3 h, thereaction mixture was washed with HCl (1M, 30 mL×4). Crude product (3.80g) was collected as a white solid and was used directly in the next stepwithout further purification. Cs₂CO₃ (1.86 g) was added into a solutionof the above crude product (3.80 g) in DMF (20 mL). The resultingsuspension was stirred at RT for 10 min before allyl bromide (1.50 mL)was added. The reaction mixture was stirred for an extra 2 h. The whiteprecipitate was filtered off with a pad of celite. The filtrate wasadded with EtOAc (40 mL) and H₂O (40 mL). Upon stirring for 10 min, theproduct precipitated. Product 9 (2.11 g) was obtained by filtration,air-dried as an off-white solid, and used in the next step withoutfurther purification.

(R)-1-(3-(4-(allyloxy)-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(10). Alcohol 9 (1.65 g) and carboxylic acid 5 (1.26 g, for synthesissee FKBD EXAMPLE 1) were dissolved in a mixture of THF (anhydrous, 5 mL)and dichloromethane (anhydrous, 10 mL). Benzoyl chloride (0.60 mL), Et3N(1.0 mL) and DMAP (18 mg) were added in order and the resultingsuspension was stirred at RT for 2 h. Without further treatment, themixture was subject to column chromatography (80-200 mesh) withEtOAc/hexane (1/2à1/1). 10 (2.50 g) was collected as a yellow foam. 1HNMR (500 MHz, CDCl3) δ 8.08 (s, 1H), 7.65 (d, J=8 Hz, 1H), 7.46 (s, 1H),7.28 (t, J=8 Hz, 1H), 7.01 (d, J=8 Hz, 1H), 6.77 (d, J=9 Hz, 1H), 6.69(d, J=5 Hz, 1H), 6.67 (s, 1H), 6.39 (dd, J=17, 1.5 Hz, 1H), 6.06 (dd,J=17, 10.5 Hz, 1H), 5.90 (ddt, J=17, 10.5, 6 Hz, 1H), 5.83 (dd, J=10.5,1.5 Hz, 1H), 5.79 (ddd, J=10.5, 8, 3.5 Hz, 1H), 5.31 (dd, J=17, 1.5 Hz,2H), 5.31 (d, J=6 Hz, 1H), 5.22 (dd, J=10.5, 1.5 Hz, 1H), 4.60 (dt, J=6,1.5 Hz, 2H), 4.33 (d, J=0.7 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.46(d, J=14 Hz, 1H), 3.09 (dd, J=18, 8 Hz, 1H), 2.78 (t, J=6 Hz, 2H), 2.70(t, J=6 Hz, 2H), 2.62-2.48 (m, 2H), 2.36 (d, J=14 Hz, 1H), 2.30-2.16 (m,1H), 2.13-2.00 (m, 1H), 1.74 (d, J=10.5 Hz, 2H), 1.62 (d, J=12 Hz, 1H),1.42 (d, J=12.6 Hz, 1H), 1.36 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 205.6,172.6, 169.8, 169.3, 166.2, 165.6, 148.9, 147.3, 140.7, 138.6, 133.5,132.0, 131.5, 129.2, 127.8, 122.0, 120.2, 119.3, 118.4, 117.2, 111.7,111.3, 76.5, 69.2, 65.5, 55.9, 55.9, 51.3, 46.8, 44.1, 38.1, 31.9, 31.1,29.3, 26.1, 25.1, 22.0, 21.9, 20.9.

4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoicacid (aFKBD). 10 (2.50 g), Pd(PPh3)4 (100 mg), N-methylaniline (1.0 mL)were mixed well in THF (20 mL) at RT for 5 h. The reaction mixture wasthen diluted with EtOAc (50 mL) and washed with HCl (1M, 50 mL×3). Theorganic phase was dried over Na2SO4, filtered and concentrated. Thecrude product was purified by column chromatography (200-400 mesh),where the byproduct can be eluted with 2% MeOH in dichloromethane,followed by the desired product with 3% MeOH and 0.05% AcOH indichloromethane. aFKBD (2.25 g) was collected as an off-white foam. ¹HNMR (500 MHz, CDCl₃) δ 8.40 (s, 1H), 7.62 (s, 1H), 7.48 (s, 1H), 7.26(t, J=7.5 Hz, 1H), 7.01 (d, J=7.5 Hz, 1H), 6.97 (dd, J=16, 7 Hz, 1H),6.86-6.74 (m, 1H), 6.74-6.58 (m, 2H), 5.85-5.68 (m, 2H), 5.39-5.24 (m,1H), 4.29 (q, J=11 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.46 (d, J=13Hz, 1H), 3.13 (t, J=13 Hz, 1H), 2.74 (d, J=5.5 Hz, 2H), 2.69 (d, J=5.5Hz, 2H), 2.63-2.48 (m, 2H), 2.36 (d, J=13 Hz, 1H), 2.30-2.15 (m, 1H),2.15-1.99 (m, 1H), 1.85 (d, J=6 Hz, 1H), 1.75 (d, J=12 Hz, 1H), 1.63 (d,J=13 Hz, 1H), 1.55-1.38 (m, 2H), 1.34 (s, 6H). ¹³C NMR (126 MHz, CDCl₃)δ 205.6, 176.8, 170.4, 169.4, 166.4, 166.1, 148.9, 147.3, 145.9, 140.7,138.5, 133.5, 129.2, 122.1, 121.9, 120.2, 119.5, 117.4, 111.8, 111.4,76.6, 69.0, 55.9, 55.8, 51.4, 46.8, 44.1, 38.1, 31.6, 31.1, 29.3, 26.2,25.0, 21.8, 20.9, 18.1. HRMS for [M+H]+ C36H44O2N11,calculated:681.3023, observed: 681.3018.

FKBD Example 22-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (eFKBD)

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(6). Alcohol 4 (3.8 g, 1.0 eq. For its synthesis see Liu et al. (2014)Angew. Chem. Int. Ed. 53:10049-55), carboxylic acid 5 (4.1 g, 1.2 eq.for synthesis see FKBD EXAMPLE 1) and DMAP (134 mg, 0.1 eq.) weredissolved in a mixture of THF (anhydrous, 35 mL) and dichloromethane(anhydrous, 35 mL) in a round bottom 42 flask under argon protection.Et3N (4.7 mL) and benzoyl chloride (2.17 mL, 2.62 g, 1.7 eq.) were addeddropwise through syringes in order and the resulting suspension wasstirred at RT for 2 h. Reaction was monitored through TLC. When fullconversion is achieved, the reaction mixture was diluted with 500 MlEtOAc, washed with 5% HCl and saturated NaHCO₃. Organic phase was washedwith brine and dried over Na₂SO₄. Then solvents were removed and productwas purified by column chromatography (80-200 mesh) with EtOAc/hexane(1/10 to 1/3). 6 (5.3 g, 69%) was collected as a light yellow foam. ¹HNMR (500 MHz, CDCl₃) δ 7.26 (d, J=8 Hz, 1H), 6.97 (d, J=8.5 Hz, 1H),6.93-6.89 (m, 1H), 6.86-6.81 (m, 1H), 6.78 (d, J=8.5 Hz, 1H), 6.71-6.64(m, 2H), 6.38 (dd, J=17, 1.5 Hz, 1H), 6.06 (dd, J=17, 10.5 Hz, 1H), 5.82(dd, J=10.5, 1.5 Hz, 1H), 5.78 (dd, J=8, 6 Hz, 1H), 5.29 (d, J=5 Hz,1H), 4.53 (s, 2H), 4.36 (d, J=11 Hz, 1H), 4.27 (d, J=11 Hz, 1H), 3.86(s, 3H), 3.84 (s, 3H), 3.48 (d, J=13 Hz, 1H), 3.17 (td, J=13, 3.0 Hz,1H), 2.67-2.44 (m, 2H), 2.37 (d, J=14 Hz, 1H), 2.32-2.18 (m, 1H),2.14-1.99 (m, 1H), 1.83-1.65 (m, 2H), 1.65-1.56 (m, 1H), 1.50-1.43 (m,2H), 1.48 (s, 9H), 1.35 (s, 3H), 1.35 (s, 3H). ¹³C NMR (126 MHz, CDCl₃)δ 204.8, 169.4, 167.8, 166.4, 165.4, 158.1, 148.9, 147.3, 141.3, 133.4,131.2, 129.7, 127.9, 120.2, 119.8, 114.2, 113.2, 111.7, 111.3, 82.3,76.7, 69.2, 65.7, 55.9, 55.8, 51.4, 46.6, 44.0, 37.9, 31.2, 28.0, 26.4,25.0, 22.1, 21.6, 21.1. HRMS for [M+H]+C38H49NO11, calculated: 696.3384,observed: 696.3386.

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (eFKBD). Compound 6 (5.3 g, 1.0 eq.) was dissolved in 60 mL ofdichloromethane in a round-bottom flask under Ar protection. Then TFA(17 mL, 11.4 g, 13 eq.) was added through a syringe in 3 portions during3.5 h while stirring at room temperature. The reaction was monitoredthrough TLC. When full conversion was achieved, solvents and TFA wereremoved under vacuum. Product was purified by column chromatography(80-200 mesh) with EtOAc/hexane (1/5à1/1). eFKBD (4.6 g, 96%) wascollected as a light yellow foam. ¹H NMR (500 MHz, CDCl₃) δ 7.28 (dd,J=3.5 Hz, 3.5 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H), 6.83-6.81 (m, 2H),6.80-6.78 (m, 1H), 6.69-6.67 (m, 2H), 6.37 (d, J=8.5 Hz, 1H), 6.05-6.02(m, 1H), 5.83-5.72 (m, 2H), 5.30-5.28 (dd, J=10, 5 Hz, 1H), 4.67 (dd,J=10, 5 Hz, 1H), 4.17 (dd, J=10, 6 Hz, 2H), 3.48-3.45 (m, 1H), 3.24-3.22(m, 1H), 2.61-2.55 (m, 2H), 2.38 (m, 1H), 2.23 (m, 1H), 2.04 (m, 1H),1.79 (m, 1H), 1.62 (m, 1H), 1.33 (m, 1H), 1.30 (m, 1H), 1.25 (s, 3H),1.24 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 204.6, 169.2, 166.7, 165.7,157.9, 149.0, 147.5, 141.7, 131.4, 129.9, 127.9, 120.0, 115.4, 111.8,111.4, 111.1, 69.3, 65.2, 60.5, 55.9, 51.7, 44.1, 38.0, 31.4, 22.1,21.1, 14.2. HRMS for [M+H]+C34H42NO11, calculated: 640.2758, observed:640.2761.

FKBD Example 34-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-morpholinopropyl)phenylamino)-4-oxobutanoicacid (Raa1)

1-(3-nitrophenyl)prop-2-en-1-one (2). Paraformaldehyde (36 g, 120 mmol)was added to a stirred solution of 1-(3-nitrophenyl)ethanone 1 (20 g,120 mmol), N-methylanilinium trifluoroacetate (26.8 g, 120 mmol) and TFA(1.4 g, 12 mmol) in THF (300 mL) at rt, the resultant reaction washeated to reflux for 16 h .The solvent was removed in vacuo, the residuewas diluted with water (100 mL) and EA (200 mL). The organic extractswere dried over Na₂SO₄ and concentrated in vacuo to afford compound 2 asa yellow solid (14.2 g, crude) used for next step directly withoutpurification. [M+H]⁺=178.1.

3-morpholino-1-(3-nitrophenyl)propan-1-one (3). To a solution of 2 (12g, 33.9 mmol, crude) in DMF (30 mL) was added Morpholine (2.95 g, 33.9mmol), followed by 4-methylbenzenesulfonic acid (5.83 g, 33.9 mmol).After stirring at room temperature for 5 h, quenched the reaction withH₂O (50 mL), extracted with EA (100 mL×3). The organic extracts weredried over Na₂SO₄ and concentrated in vacuo to give a crude productwhich was further purified by column (SiO₂, Methanol/DCM=0-10% aseluent) to afford compound 3 (6.6 g, 74%) as a yellow oil. [M+H]⁺=265.2

1-(3-aminophenyl)-3-morpholinopropan-1-one (4). To a solution of 3 (4.2g, 15.9 mmol) in THF (20 mL) was added 10% Pd/C (wet, 840 mg) at rt. Theresulting reaction mixture was hydrogenated with H₂ (g) at rt for 8 h.The reaction mixture was then filtered and concentrated in vacuo toafford crude compound 4 (3.46 g, crude) as a yellow oil used for nextstep directly. [M+H]⁺=235.1

tert-butyl 4-(3-(3-morpholinopropanoyl)phenylamino)-4-oxobutanoate (6).To a solution of 4 (5.05 g, 21.5 mmol) and 4-tert-butoxy-4-oxobutanoicacid 5 (4.86 g, 27.95 mmol) in DMF (20 mL) was added DIPEA (5.55 g, 43mmol) followed by HATU (10.62 g, 27.95 mmol) at rt. The resultingreaction mixture was stirred at rt for 2 h. Quenched the reaction withH₂O (50 mL), extracted with EA (100 mL×3). The organic extracts weredried over Na₂SO₄ and concentrated in vacuo to give a crude productwhich was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent)to afford compound 6 (4.3 g, 51%) as a yellow solid. [M+H]⁺=391.0

(R)-tert-butyl4-(3-(1-hydroxy-3-morpholinopropyl)phenylamino)-4-oxobutanoate (7). To asolution of ketone 6 (4.1 g, 10.5 mmol) in anhydrous THF (40 mL) wasadded (+)DIPChloride (42 mmol) in heptane (1.7 M, 24.7 mL) at −20° C.The resulting reaction mixture was stirred at −20° C. until completeconversion of 6, the quenched with2,2′-(ethane-1,2-diylbis(oxy))diethanamine (7 g, 47.25 mmol) by formingan insoluble complex. After stirring at rt for another 30 min, thesuspension was filtered through a pad of celite and concentrated invacuo to give a crude product which was further purified by column(SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 7 (1.0 g, 24%) as anoff white solid. [M+H]⁺=393.0

(S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-morpholinopropyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (1.0 g, 2.55 mmol) and 8 (952 mg, 3.06 mmol) inanhydrous DCM (25 mL) was cooled to −20° C. before a solution of DCC(630 mg, 3.06 mmol) in anhydrous DCM (2 mL) was added, followed by theaddition of a solution of 4-(dimethylamino)pyridine (DMAP, 31 mg, 0.255mmol) under argon atmosphere. The resulting white suspension was stirredat −20° C. for 2 h. The reaction mixture was then filtered and thefiltrate were dried over Na₂SO₄ and concentrated in vacuo to give acrude product which was further purified by column (SiO₂, CH₃OH/DCM=0-5%as eluent) to afford compound 9 (1.3 g, 76%) as a white solid.[M+H]⁺=686.0

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-morpholinopropyl)phenylamino)-4-oxobutanoicacid (RAa-1). To a solution of 9 (1.3 g, 1.9 mmol) in DCM (10 mL) wasadded TFA (2 mL) at rt. The resulting mixture was stirred at rt for 3 h.The reaction mixture was charged to silica-gel flash column directly(CH₃OH/DCM=0-5% as eluent) to afford RAa-1 as a white solid (620 mg,51%).

FKBD Example 44-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-morpholinobutyl)phenylamino)-4-oxobutanoicacid (Raa2)

3-(3-nitrobenzoyl)-dihydrofuran-2(3H)-one (3). To a stirred solution ofdihydrofuran-2(3H)-one 2 (6.02 g, 70 mmol) in anhydrous THF (60 mL) wasadded LiHMDS (1M in THF, 77 mL, 77 mmol) at −78° C. and stirred for 2 hunder argon atmosphere. Then the solution of 3-nitrobenzoyl chloride 1(6.5 g, 35 mmol) in anhydrous THF (10 mL) was added at −78° C. Theresultant reaction mixture was slowly warmed to rt and stirred at rt for16 h. Quenched the reaction with saturated NH₄Cl_(aq) (20 mL), extractedwith EA (100 mL×3). The organic extracts were dried over Na₂SO₄ andconcentrated in vacuo to afford compound 3 (8.5 g, crude) as a yellowoil used for next step directly without purification. [M+H]⁺=236.1

4-bromo-1-(3-nitrophenyl)butan-1-one (4). A solution of 3 (25.9 g, 110mmol, crude) in 40% HBr (150 mL) was heated to 70° C. for 2 h. Thereaction mixture was cooled to rt and adjusted the pH to 5-6 withsaturated NaHCO_(3 aq), extracted with EA (200 mL×3). The organicextracts were dried over Na₂SO₄ and concentrated in vacuo to give acrude product which was further purified by column (SiO₂, EA/PE=0-10% aseluent) to afford compound 4 (18.5 g, 74% for 2 steps) as a yellow oil.

4-morpholino-1-(3-nitrophenyl)butan-1-one (5). To a solution of 4 (8.5g, 31.25 mmol) and Morpholine (2.72 g, 31.25 mmol) in CH₃CN (100 mL) wasadded K₂CO₃ (8.64 g, 62.5 mmol) at rt. The resulting reaction mixturewas heated to reflux for 2 h. The reaction mixture was then filtered andconcentrated in vacuo to give a crude product which was further purifiedby column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 5 (4.6g, 53%) as a yellow oil. [M+H]⁺=279.2

1-(3-aminophenyl)-4-morpholinobutan-1-one (6). A solution of 5 (5.9 g,21.2 mmol) in THF (60 mL) was added 10% Pd/C (wet, 1.18 g) at rt. Theresulting reaction mixture was hydrogenated with H₂ (g) at rt for 10 h.The reaction mixture was then filtered and concentrated in vacuo toafford crude compound 6 (4.8 g, crude) as a yellow solid used for nextstep directly. [M+H]⁺=249.0

To a solution of 6 (4.8 g, 19.35 mmol) and 4-tert-butoxy-4-oxobutanoicacid 7 (4.86 g, 27.95 mmol) in DMF (15 mL) was added DIPEA (5.0 g, 38.7mmol) followed by HATU (9.56 g, 25.15 mmol) at rt. The resultingreaction mixture was stirred at rt for 2 h. Quenched the reaction withH₂O (50 mL), extracted with EA (100 mL×3). The organic extracts weredried over Na₂SO₄ and concentrated in vacuo to give a crude productwhich was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent)to afford compound 8 (6.6 g, 84%) as a yellow solid. [M+H]⁺=405.0

(R)-tert-butyl4-(3-(1-hydroxy-4-morpholinobutyl)phenylamino)-4-oxobutanoate (9). To asolution of ketone 8 (5.0 g, 12.4 mmol) in anhydrous THF (20 mL) wasadded (+)DIPChloride (49.6 mmol) in heptane (1.7 M, 29 mL) at −20° C.The resulting reaction mixture was stirred at −20° C. until completeconversion of 8, then quenched with2,2′-(ethane-1,2-diylbis(oxy))diethanamine (8.3 g, 55.8 mmol) by formingan insoluble complex. After stirring at rt for another 30 min, thesuspension was filtered through a pad of celite and concentrated invacuo to give a crude product which was further purified by column(SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 9 as an off whitesolid (2.5 g, 50%). [M+H]⁺=407.3

(S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-4-morpholinobutyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(11). A solution of 9 (2.45 g, 6.05 mmol) and 10 (2.29 g, 7.38 mmol) inanhydrous DCM (40 mL) was cooled to −20° C. before a solution of DCC(1.52 g, 7.38 mmol) in anhydrous DCM (5 mL) was added, followed by theaddition of a solution of 4-(dimethylamino)pyridine (DMAP, 75 mg, 0.615mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resultingwhite suspension was stirred at −20° C. for 2 h. The reaction mixturewas then filtered and the filtrate were dried over Na₂SO₄ andconcentrated in vacuo to give a crude product which was further purifiedby column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 11 as awhite solid (3 g, 69%). [M+H]⁺=700.0

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-morpholinobutyl)phenylamino)-4-oxobutanoicacid (Raa2). To a solution of 11(1.0 g, 1.42 mmol) in DCM (10 mL) wasadded TFA (2 mL) at rt. The resulting mixture was stirred at rt for 2 h.The reaction mixture was charged to silica-gel flash column directly(CH₃OH/DCM=0-5% as eluent) to afford Raa2 (550 mg, 60%) as a whitesolid.

FKBD Example 54-(3-((R)-3-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)propyl)phenylamino)-4-oxobutanoicacid (Raa3)

tert-butyl 4-(3-(3-nitrophenyl)-3-oxopropyl)piperazine-1-carboxylate(3). To a solution of 1(10 g, 28.2 mmol, crude) in DMF (20 mL) was addedDIPEA (3.64 g, 28.2 mmol), followed by 2 (5.24 g, 28.2 mmol). Afterstirring at room temperature for 2 h, quenched the reaction with H₂O(100 mL), extracted with EA (100 mL×3). The organic extracts were driedover Na₂SO₄ and concentrated in vacuo to give a crude product which wasfurther purified by column (SiO₂, Methanol/DCM=0-10% as eluent) toafford compound 3 as a yellow oil (6.1 g, 60%). [M+H]⁺=364.2

tert-butyl 4-(3-(3-aminophenyl)-3-oxopropyl)piperazine-1-carboxylate(4). A solution of 3 (6.1 g, 15.9 mmol) in THF (50 mL) was added 10%Pd/C (wet, 1.22 g) at rt. The resulting reaction mixture washydrogenated with H₂ (g) at rt for 8 h. The reaction mixture was thenfiltered and concentrated in vacuo to afford crude compound 4 as a brownsolid (5.5 g, crude) used for next step directly. [M+H]⁺=334.3

tert-butyl4-(3-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-oxopropyl)piperazine-1-carboxylate(6). To a solution of 4 (5.2 g, 15.6 mmol) and4-tert-butoxy-4-oxobutanoic acid 5 (3.53 g, 20.27 mmol) in DMF (35 mL)was added DIPEA (5.04 g, 38.99 mmol) followed by HATU (7.71 g, 20.27mmol) at rt. The resulting reaction mixture was stirred at rt for 4 h.Quenched the reaction with H₂O (50 mL), extracted with EA (100 mL×3).The organic extracts were dried over Na₂SO₄ and concentrated in vacuo togive a crude product which was further purified by column (SiO₂,PE/EA=0-50% as eluent) to afford compound 6 (4.3 g, 56%) as a yellowsolid. [M+H]⁺=490.4

(R)-tert-butyl4-(3-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-hydroxypropyl)piperazine-1-carboxylate(7). To a solution of ketone 6 (3.8 g, 7.76 mmol) in anhydrous THF (30mL) was added (+)DIPChloride (38.8 mmol) in heptane (1.7 M, 23 mL) at−20° C. The resulting reaction mixture was stirred at −20° C. untilcomplete conversion of 6, the quenched with2,2′-(ethane-1,2-diylbis(oxy))diethanamine (6.32 g, 42.68 mmol) byforming an insoluble complex. After stirring at rt for another 30 min,the suspension was filtered through a pad of celite and concentrated invacuo to give a crude product which was further purified by column(SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 7 as an off whitesolid (1.9 g, 51%). [M+H]⁺=492.3

tert-butyl4-((R)-3-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)propyl)piperazine-1-carboxylate(9). A solution of 7 (1.03 g, 2.1 mmol) and 8 (784 mg, 2.52 mmol) inanhydrous DCM (20 mL) was cooled to −20° C. before a solution of DCC(865 mg, 4.2 mmol) in anhydrous DCM (2 mL) was added, followed by theaddition of a solution of 4-(dimethylamino)pyridine (DMAP, 26 mg, 0.21mmol) under argon atmosphere. The resulting white suspension was stirredat −20° C. for 2 h. The reaction mixture was then filtered and thefiltrate were dried over Na₂SO₄ and concentrated in vacuo to give acrude product which was further purified by column (SiO₂, CH₃OH/DCM=0-5%as eluent) to afford compound 9 as a yellow solid (1.2 g, 72%).[M+H]⁺=784.9

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(piperazin-1-yl)propyl)phenylamino)-4-oxobutanoicacid (10). To a solution of 9 (1.2 g, 1.9 mmol) in DCM (6 mL) was addedTFA (3 mL) at rt. The resulting mixture was stirred at rt for 3 h. Thereaction mixture was concentrated in vacuo to afford compound 10 (1.1 g,crude) as a yellow solid. [M+H]⁺=628.9

4-(3-((R)-3-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)propyl)phenylamino)-4-oxobutanoicacid (Raa3). To a solution of 10 (1.1 g, 1.74 mmol) in DMF (4 mL) wasadded Na₂CO₃ (369 mg, 3.48 mmol) followed by FmocChloride (450 mg, 1.74mmol) at rt. The resulting reaction mixture was stirred at rt for 30min. Quenched the reaction with H₂O (10 mL), extracted with EA (30mL×3). The organic extracts were dried over Na₂SO₄ and concentrated invacuo to give a crude product which was further purified by column(SiO₂, Methanol/DCM=0-5% as eluent) to afford Raa3 (680 mg, 46%) as awhite solid.

FKBD Example 64-(3-((R)-4-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)butyl)phenylamino)-4-oxobutanoicacid (Raa4)

tert-butyl 4-(4-(3-nitrophenyl)-4-oxobutyl)piperazine-1-carboxylate (3).To a solution of 1(10.5 g, 38.6 mmol) and 2 (7.2 g, 38.6 mmol) in CH₃CN(100 mL) was added K₂CO₃ (10.7 g, 77.2 mmol) at rt. The resultingreaction mixture was heated to reflux for 2 h. The reaction mixture wasthen filtered and concentrated in vacuo to give a crude product whichwas further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) toafford compound 3 (8.3 g, 57%) as a yellow solid. [M+H]⁺=378.0

tert-butyl 4-(4-(3-aminophenyl)-4-oxobutyl)piperazine-1-carboxylate (4).A solution of 3 (8.3 g, 22 mmol) in THF (60 mL) was added 10% Pd/C (wet,1.66 g) at rt. The resulting reaction mixture was hydrogenated with H₂(g) at rt for 10 h. The reaction mixture was then filtered andconcentrated in vacuo to afford crude compound 4 (7.4 g, crude) as ayellow solid used for next step directly. [M+H]⁺=348.3

tert-butyl 4-(3-(4-morpholinobutanoyl)phenylamino)-4-oxobutanoate (6).To a solution of 4 (7.4 g, 21.3 mmol) and 4-tert-butoxy-4-oxobutanoicacid 5 (4.82 g, 27.6 mmol) in DMF (15 mL) was added DIPEA (5.5 g, 42.6mmol) followed by HATU (10.5 g, 27.69 mmol) at rt. The resultingreaction mixture was stirred at rt for 2 h. Quenched the reaction withH₂O (50 mL), extracted with EA (100 mL×3). The organic extracts weredried over Na₂SO₄ and concentrated in vacuo to give a crude productwhich was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent)to afford compound 6 (8.5 g, 79%) as a yellow solid. [M+H]⁺=504.0

(R)-tert-butyl4-(4-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-4-hydroxybutyl)piperazine-1-carboxylate(7). To a solution of ketone 6 (4.5 g, 8.9 mmol) in anhydrous THF (20mL) was added (+)DIPChloride (35.6 mmol) in heptane (1.7 M, 21 mL) at−20° C. The resulting reaction mixture was stirred at −20° C. untilcomplete conversion of 6, then quenched with2,2′-(ethane-1,2-diylbis(oxy))diethanamine (5.9 g, 40.0 mmol) by formingan insoluble complex. After stirring at rt for another 30 min, thesuspension was filtered through a pad of celite and concentrated invacuo to give a crude product which was further purified by column(SiO₂, Methanol/EA=0-5% as eluent) to afford compound 7 as an off whitesolid (2.5 g, 55%). [M+H]⁺=506.0

tert-butyl4-((R)-4-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)butyl)piperazine-1-carboxylate(9). A solution of 7 (2.3 g, 4.5 mmol) and 8 (1.68 g, 5.4 mmol) inanhydrous DCM (30 mL) was cooled to −20° C. before a solution of DCC(1.11 g, 5.4 mmol) in anhydrous DCM (5 mL) was added, followed by theaddition of a solution of 4-(dimethylamino)pyridine (DMAP, 55 mg, 0.615mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resultingwhite suspension was stirred at −20° C. for 2 h. The reaction mixturewas then filtered and the filtrate were dried over Na₂SO₄ andconcentrated in vacuo to give a crude product which was further purifiedby column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 9 as awhite solid (2.9 g, 80%). [M+H]⁺=799.5

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-(piperazin-1-yl)butyl)phenylamino)-4-oxobutanoicacid (10). To a solution of 9 (2.9 g, 3.6 mmol) in DCM (10 mL) was addedTFA (3 mL) at rt. The resulting mixture was stirred at rt for 4 h. Thereaction mixture was charged to silica-gel flash column directly(CH₃OH/DCM=0-5% as eluent) to afford compound 10 (2.6 g, crude) as ayellow solid used for next step directly. [M+H]⁺=643.4

4-(3-((R)-4-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)butyl)phenylamino)-4-oxobutanoicacid (Raa4). To a solution of 10 (1.2 g, 1.62 mmol) in DMF (2 mL) wasadded Na₂CO₃ (343 mg, 3.24 mmol) followed by FmocChloride (419 mg, 1.62mmol) at rt. The resulting reaction mixture was stirred at rt for 30min. Quenched the reaction with H₂O (10 mL), extracted with EA (30mL×3). The organic extracts were dried over Na₂SO₄ and concentrated invacuo to give a crude product which was further purified by column(SiO₂, Methanol/DCM=0-5% as eluent) to afford Raa4 (570 mg, 40%) as awhite solid.

FKBD Example 74-(5-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-ylamino)-4-oxobutanoicacid (Raa5)

1-(5-aminopyridin-3-yl)ethanone (2). To a solution of 1(13 g, 78.3 mmol)in THF (100 mL) was added 10% Pd/C (wet, 8.0 g) at rt. The resultingreaction mixture was stirred at rt for 10 h under H₂ (g). The reactionmixture was then filtered and concentrated in vacuo to give a crudeproduct which was further purified by column (SiO₂, Methanol/DCM=0-5% aseluent) to afford compound 2 (10 g, 94%) as a yellow solid. [M+H]⁺=137.0

(E)-1-(5-aminopyridin-3-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (4).To a solution of 2 (6.5 g, 47.8 mmol) and 3 (7.9 g, 47.8 mmol) in CH₃OH(60 mL) was added LiOH.H₂O (2 g, 47.8 mmol) at 0° C. The resultingreaction mixture was stirred at rt for 3 h. The solvent was removed invacuo and the residue was diluted with DCM and H₂O. The organic extractswere dried over Na₂SO₄ and concentrated in vacuo to give a crude productwhich was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent)to afford compound 4 (1.8 g, 13%) as a yellow solid. [M+H]⁺=285.0

(E)-tert-butyl4-(5-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-3-ylamino)-4-oxobutanoate(6). To a solution of 4 (1.8 g, 6.3 mmol) and4-tert-butoxy-4-oxobutanoic acid 5 (1.1 g, 6.3 mmol) in DCM (35 mL) wasadded Et₃N (12.7 g, 12.6 mmol) followed by T₃P (50% in EtOAc, 8.0 g,12.6 mmol) at rt. The resulting reaction mixture was stirred at rt for 1h. Quenched the reaction with H₂O (20 mL), extracted with DCM (40 mL×2).The organic extracts were dried over Na₂SO₄ and concentrated in vacuo togive a crude product which was further purified by column (SiO₂,Methanol/DCM=0-5% as eluent) to afford compound 6 (1.88 g, 68%) as ayellow solid. [M+H]⁺=440.9

tert-butyl4-(5-(3-(3,4-dimethoxyphenyl)propanoyl)pyridin-3-ylamino)-4-oxobutanoate(7). A solution of 6 (1.88 g, 4.27 mmol) in THF (50 mL) and Methanol (5mL) was added 10% Pd/C (wet, 380 mg) at rt. The resulting reactionmixture was hydrogenated with H₂ (g) at rt for 4 h. The reaction mixturewas then filtered and concentrated in vacuo to give a crude productwhich was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent)to afford compound 7 (1.34 g, 71%) as a brown solid. [M+H]⁺=442.9

(R)-tert-butyl4-(5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-3-ylamino)-4-oxobutanoate(8). To a solution of ketone 7 (1.34 g, 3.0 mmol) in anhydrous THF (20mL) was added (+)DIPChloride (12.0 mmol) in heptane (1.7 M, 7.05 mL) at−20° C. The resulting reaction mixture was stirred at −20° C. untilcomplete conversion of 7, then quenched with2,2′-(ethane-1,2-diylbis(oxy))diethanamine (2.0 g, 13.5 mmol) by formingan insoluble complex. After stirring at rt for another 30 min, thesuspension was filtered through a pad of celite and concentrated invacuo to give a crude product which was further purified by column(SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 8 (0.99 g, 74%) as awhite solid. [M+H]⁺=445.0

(S)—((R)-1-(5-(4-tert-butoxy-4-oxobutanamido)pyridin-3-yl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(10). A solution of 8 (990 mg, 2.22 mmol) and 9 (827 mg, 2.66 mmol) inanhydrous DCM (20 mL) was cooled to −20° C. before a solution of DCC(548 mg, 2.66 mmol) in anhydrous DCM (2 mL) was added, followed by theaddition of a solution of 4-(dimethylamino)pyridine (DMAP, 27 mg, 0.22mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resultingwhite suspension was stirred at −20° C. for 2 h. The reaction mixturewas then filtered and the filtrate were dried over Na₂SO₄ andconcentrated in vacuo to give a crude product which was further purifiedby column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 10 (1.3 g,79%) as a white solid. [M+H]⁺=738.0

4-(5-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-ylamino)-4-oxobutanoicacid (Raa5). To a solution of 10 (1.3 g, 1.76 mmol) in DCM (10 mL) wasadded TFA (5 mL) at rt. The resulting mixture was stirred at rt for 2 h.The reaction mixture was charged to silica-gel flash column directly(CH₃OH/DCM=0-5% as eluent) to afford Raa5 (960 mg, 80%) as a whitesolid.

FKBD Example 84-(6-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-2-ylamino)-4-oxobutanoicacid (Raa6)

(E)-1-(6-aminopyridin-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3).To a solution of 1(3.75 g, 27.57 mmol) and 2 (4.58 g, 27.57 mmol) inCH₃OH (40 mL) was added LiOH.H₂O (1.74 g, 41.35 mmol) at rt. Theresulting reaction mixture was stirred at rt for 3 h. The solvent wasremoved in vacuo and the residue was diluted with DCM and H₂O. Theorganic extracts were dried over Na₂SO₄ and concentrated in vacuo togive a crude product which was further purified by column (SiO₂,Methanol/DCM=0-5% as eluent) to afford compound 3 (3.2 g, 41%) as ayellow solid. [M+H]⁺=285.0

(E)-tert-butyl4-(6-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-2-ylamino)-4-oxobutanoate(5). To a solution of 3 (3.2 g, 11.26 mmol) and4-tert-butoxy-4-oxobutanoic acid 4 (2.35 g, 13.5 mmol) in Pyridine (10mL) was added POCl₃ (2.58 g, 16.89 mmol) at 0° C. The resulting reactionmixture was stirred at 0° C. for 15 min. Quenched the reaction with H₂O(20 mL), extracted with EA (30 mL×3). The organic extracts were driedover Na₂SO₄ and concentrated in vacuo to give a crude product which wasfurther purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to affordcompound 5 (2.45 g, 49%) as a yellow solid. [M+H]⁺=440.9

tert-butyl4-(6-(3-(3,4-dimethoxyphenyl)propanoyl)pyridin-2-ylamino)-4-oxobutanoate(6). A solution of 5 (2.45 g, 5.56 mmol) in THF (30 mL) was added 10%Pd/C (wet, 500 mg) at rt. The resulting reaction mixture washydrogenated with H₂ (g) at rt for 4 h. The reaction mixture was thenfiltered and concentrated in vacuo to give a crude product which wasfurther purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to affordcompound 6 (1.5 g, 61%) as a yellow solid. [M+H]⁺=443.3

(R)-tert-butyl4-(6-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-2-ylamino)-4-oxobutanoate(7). To a solution of ketone 6 (1.4 g, 3.16 mmol) in anhydrous DCM (20mL) was added (+)DIPChloride (12.64 mmol) in heptane (1.7 M, 7.5 mL) at−20° C. The resulting reaction mixture was stirred at −20° C. untilcomplete conversion of 7, then quenched with2,2′-(ethane-1,2-diylbis(oxy))diethanamine (2.1 g, 14.22 mmol) byforming an insoluble complex. After stirring at rt for another 30 min,the suspension was filtered through a pad of celite and concentrated invacuo to give a crude product which was further purified by column(SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 7 (1.0 g, 71%) as awhite solid. [M+H]⁺=445.3

(S)—((R)-1-(6-(4-tert-butoxy-4-oxobutanamido)pyridin-2-yl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (1.0 g, 2.24 mmol) and 8 (836 mg, 2.69 mmol) inanhydrous DCM (20 mL) was cooled to −20° C. before a solution of DCC(554 mg, 2.69 mmol) in anhydrous DCM (2 mL) was added, followed by theaddition of a solution of 4-(dimethylamino)pyridine (DMAP, 27 mg, 0.22mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resultingwhite suspension was stirred at −20° C. for 2 h. The reaction mixturewas then filtered and the filtrate were dried over Na₂SO₄ andconcentrated in vacuo to give a crude product which was further purifiedby column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 9 (0.38 g,23%) as a white solid. [M+H]⁺=738.4

4-(6-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-2-ylamino)-4-oxobutanoicacid (Raa6). To a solution of 9 (0.38 g, 1.76 mmol) in DCM (5 mL) wasadded TFA (2 mL) at rt. The resulting mixture was stirred at rt for 2 h.The reaction mixture was charged to silica-gel flash column directly(CH₃OH/DCM=0-5% as eluent) to afford Raa6 (310 mg, 89%) as a whitesolid.

FKBD Example 94-((6-((R)-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyrazin-2-yl)amino)-4-oxobutanoicacid (Raa7)

6-(1-butoxyvinyl)pyrazin-2-amine (3). To a solution of 1(16 g, 124 mmol)in ethylene glycol (150 mL) was added Pd(AcO)₂ (0.8 g, 3.7 mmol) andDPPF (4.12 g, 7.4 mmol) at rt. Degassed by Ar2, and then 2 and Et₃N wasinjected sequentially. The reaction mixture was heated to reflux andreacted for 1.5 h. The product mixture was poured into water (300 ml),extracted with DCM (100 ml*3). Combined the organic phase and washedwith brine (100 ml*3). Filtered and concentrated to get 3 (12 g, 50%) aswhite solid. [M+H]⁺=194

1-(6-aminopyrazin-2-yl)ethan-1-one (4). To a solution of 3 (12 g, 62mmol) in DCM (50 ml) was added 5% HCl (20 ml). The reaction mixture wasstirred at rt for 0.5 h. Poured the product mixture into water (200 ml),adjusted pH to 8-9 with K₂CO₃ (aq). Extracted with DCM (50 ml*6),combined the organic phase and concentrated to get the crude. Purifiedby silca gel chromatography (PE/EA=20-30% as eluent) to give product 4(2.9 g, 34%) as yellow solid. [M+H]⁺=138

(E)-1-(5-amiopyrazin-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6).To a solution of 4 (2.9 g, 21 mmol) in MeOH (20 ml) was added LiOH (1.74g, 42 mol) and 5 (3.43 g, 21 mmol). The reaction mixture was stirred at40° C. for 1 h. Poured the product mixture into water (200 ml), filtereduntil no more precipitation, washed the solid cake with water, and thenlittle MeOH. Dried to get product 6 (3.8 g, 64.5%) as yellow solid.[M+H]⁺=286

tert-butyl(E)-4-((6-(3-(3,4-dimethoxyphenyl)acryloyl)pyrazin-2-yl)amino-4-oxobutanoate(8). To a solution of 8 (3.8 g, 133 mmol) and 7 (4.64 g, 266 mmol) inpyridine (100 ml) was added POCl₃ (6.12 g, 400 mmol) at 0° C. Thereaction mixture was stirred at 0° C. for 30 min. Poured the productmixture into water (300 ml), extracted with DCM (100 ml*3), combined theorganic phase and washed with brine (100 ml*5). Dried over Na₂SO₄,filtered and concentrated to get the crude. Purified by silca gelchromatography (MeOH/DCM=1-2% as eluent) to give product 8 (5 g, 68%) asyellow solid. [M+H]⁺=442

tert-butyl4-((6-(3-(3,4-dimethoxyphenyl)propannoyl)pyrazin-2-yl)amino)-4-oxobutanoate(9). To a solution of 8 (5.0 g, 113 mmol) in THF was added Pd/C (500 mg,10%), the reaction mixture was degassed with H₂*5, stirred at rt for 4h. Filtered and concentrated the filtrate to get the crude. Purified bysilca gel chromatography (MeOH/DCM=1-2% as eluent) to give product 9(2.0 g, 40%) as yellow solid. [M+H]⁺=444

tert-butyl(R)4-((6-(3-(3,4-dimethoxyphenyl)-1-hydroxyphenyl)pyrazin-2-yl)amino)-4-oxobutanoate(11). To a solution of 9 (2.0 g, 45 mmol) in DCM (50 ml) was added DIPCl(14.5 g, 450 mmol) at −20° C., degassed with Ar₂. The reaction mixturewas stirred at −20° C. for 5 h. Quenched with 10 (6.75 g, 455 mmol). Theproduct mixture was concentrated directly, and the brown residue waspurified by silca gel chromatography (MeOH/DCM=2-5% as eluent) to giveproduct 11 (1.0 g, 50%) as yellow solid. [M+H]⁺=446

(R)-1-(6-(4-tert-butoxy)-4-oxobutanamido)pyrazin-2-yl)-3-(3,4-dimethoxyphenyl(S)-1(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(13). To a solution of 11 (1.0 g, 22 mmol) in DCM (30 ml) was added 12(1.05 g, 34 mmol) at −20° C., and degassed with Ar₂, then DCC (0.7 g, 34mmol) and DMAP (0.03 g, 2.2 mmol) in DCM was injected sequentially. Thereaction mixture was stirred at −20° C. for 1 h. Filtered and washed thesolid cake with DCM (20 ml), the filtrate was combined and evaporated toget the crude. Purified by silca gel chromatography (MeOH/DCM=1-2% aseluent) to give product 13 (1.8 g, 85%) as yellow solid. [M+H]⁺=739

4-((6-((R)—(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyrazin-2-yl)amino)-4-oxobutanoicacid (Raa7). To a solution of 13 (1.8 g, 24 mmol) in DCM (20 ml) wasadded TFA (20 ml). The reaction mixture was stirred at rt for 2 h.Concentrated the product mixture directly, the yellow residue waspurified by silca gel chromatography (MeOH/DCM=1-2% as eluent) to giveproduct Raa7 (500 mg, 30%) as light yellow solid.

FKBD Example 10 4-((3-((R)-1-(((S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)morpholine-3-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoicacid (Raa8)

(E)-3-(3,4-dimethoxyphenyl)-1-(3-nitrophenyl)prop-2-en-1-one (3). To thesolution of 3,4-dimethoxybenzaldehyde 1 (60 g, 360 mmol) and1-(3-nitrophenyl)ethan-1-one 2 (59.6 g, 360 mmol) in MeOH (1100 mL) wasadded NaOH (15 g) at 0° C. The resulting solution was stirred at rt for10 h. The precipitate was collected to give compound 3 as a yellow solid(97 g, 86%). [M+Na]⁺=336.1

1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propan-1-one (4). A solution of3 (32 g, 110 mmol) and 10% Pd/C (10 g) in THF (120 mL) was hydrogenatedwith H₂ for 8 h at room temperature. The reaction mixture was thenfiltered and concentrated. The residue was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:3) to give compound 4 as a white solid(24 g, 76%). [M+H]⁺=286.2

tert-butyl4-((3-(3-(3,4-dimethoxyphenyl)propanoyl)phenyl)amino)-4-oxobutanoate(5). To a solution of 4 (12.0 g, 42 mmol) in DCM (30 mL) was added4-tert-butoxy-4-oxobutanoic acid (8.8 g, 50 mmol), DIPEA (13.6 g, 105mmol) and HATU(19.2 g, 50 mmol). The mixture was stirred at rt for 16 h.The product was purified by silica-gel flash column chromatography(AcOEt/PE 1:2) to give compound 5 as a white solid (16 g, 79%).[M+Na]⁺=464.0

tert-butyl(R)-4-((3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenyl)amino)-4-oxobutanoate(6). A solution of ketone 5 (11.9 g, 26.9 mmol) in dry THF (120 mL) at−20° C. was treated with a solution of (+)-DIPChloride (135 mmol) inheptane (1.7 M, 79 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 5, then quenched with2,2′-(ethylenedioxy)diethylamine (20 g) by forming an insoluble complex.After stirring at RT for another 30 min, the suspension was filteredthrough a pad of celite and concentrated. The crude compound waspurified by silica-gel flash column chromatography (AcOEt/PE 3:1) togive compound 6 as a light yellow oil (7.9 g, 66%, ee 97%).[M+Na]⁺=466.3

(R)-1-(3-(4-(tert-butoxy)-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)morpholine-3-carboxylate(8). A solution of 6 (2.36 g, 5.32 mmol) and 7 (2 g, 6.38 mmol) inCH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (1.65 g,7.98 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 65 mg, 0.53 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 2:1) to give compound 8 as a lightyellow oil (2.5 g, 64%). [M+Na]⁺=761.4

4-((3-((R)-1-(((S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)morpholine-3-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoicacid (Raab). A solution of 8 (2.5 g, 3.45 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Raab (815 mg, 38%) as a paleyellow solid.

FKBD Example 114-((3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoicacid (Raa9)

1-((9H-fluoren-9-yl)methyl)3-((R)-1-(3-(4-(tert-butoxy)-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl)(S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-1,3-dicarboxylate(3). A solution of 1(1.35 g, 3.04 mmol) and 2 (1.95 g, 3.65 mmol) inCH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (940 mg,4.56 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 37 mg, 0.3 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 3 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (DCM/MeOH 96:4) to give compound 3 as awhite solid (3.0 g, quant.). [M+Na]⁺=981.6

4-((3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoicacid (Raa9). A solution of 3 (1.5 g, 1.56 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Raa9 (1.4 g, 99%) as a whitesolid.

FKBD Example 12(S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carboxylate (Raa10)

(S)-1-(9H-fluoren-9-yl)methyl3-((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl)4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-1,3-dicarboxylate(2). To the solution of 1(1.3 g, 1.35 mmol) in DMF (5 mL) was added TBAF(3.2 ml, 1.0 M, 3.18 mmol) at 0° C. The resulting solution was heated toroom temperature for 5 h. After this time the reaction mixture waswashed with NaHCO₃ (aq., 50 ml*3) and NaCl (aq., 50 ml*3). The organicphase was concentracted. The reaction mixture was purified on silicawith DCM/MEOH=50/1 to give 2 (800 mg,80%) as a colourless oil.[M+H]⁺=738.4

(S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carboxylate(Raa10). A solution of 2 (800 mg, 1.08 mmol) in CHOOH (1.6 mL) wastreated with an aqueous solution of formaldehyde (37% in water, 0.8 ml,1.3 mmol) and allowed to stir at 50° C. for 1 h. After this time thereaction mixture was purified with DCM/MeOH=100/1 give 3 (400 mg, 50%)as a colorless oil. [M+H]⁺=751.9

FKBD Example 13(S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-3-hydroxy-4-oxobutanoicacid (Raa11)

tert-butyl 3-(3-(3,4-dimethoxyphenyl)propanoyl)phenylcarbamate (2). Tothe solution of 1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propan-1-one 1(8.5 g, 29.79 mmol) in 1,4-dioxane (85 mL) was added (Boc)₂O (9.75 g,44.68 mmol). The resulting solution was heated to 100° C. for 3 h. Thesolvent was evaporated and the residue (10.3 g, crude) was used directlyfor the next step without purification. [M+Na]⁺=408

(R)-tert-butyl3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylcarbamate (3). Asolution of ketone 2 (10 g, crude) in dry THF (200 mL) at −20° C. wastreated with a solution of (+)-DIPChloride in heptane (1.7 M, 76.2 mL)at −20° C. The resulting mixture was reacted at −20° C. until completeconversion of 2, then quenched with 2,2′-(ethylenedioxy)diethylamine(23.1 g) by forming an insoluble complex. After stirring at RT foranother 30 min, the suspension was filtered through a pad of celite andconcentrated. The crude compound was purified by silica-gel flash columnchromatography (AcOEt/PE 1:3) to give compound 3 as a light yellow oil(8.3 g, 80%). [M+Na]⁺=410

(S)—((R)-1-(3-(tert-butoxycarbonylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(5). A solution of 3 (8.3 g, 21.42 mmol) and 4 (8 g, 25.7 mmol) inCH₂Cl₂ (100 mL) was cooled to −20° C. before a solution of DCC (5.3 g,25.7 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 318 mg, 2.6 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 5 as a lightyellow oil (12 g, 83%). [M+Na]⁺=703.3

(S)—((R)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(6). To a solution of 5 (5 g, 7.34 mmol) in DCM (30 ml) was added TFA (6ml). The mixture was stirred at 35° C. for 6 h. The solvent wasevaporated and the residue (5.0 g, crude) was used directly for the nextstep without purification. [M+H]⁺=580.8

(S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-3-hydroxy-4-oxobutanoicacid (Raa11). A solution of 6 (1.0 g, crude) in DCM (20 mL) was added 7(400 mg, 3.4 mmol) and DMAP (25 mg, 0.2 mmol). The mixture was allowedto react at room temperature until complete conversion. The reactionmixture was charged to silica-gel flash column directly (DCM/MeOH=10:1)to afford Raa11 (450 mg, 38%) as a white solid.

FKBD Example 14(S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-2-hydroxy-4-oxobutanoicacid (Raa12)

The synthesis of 6 is the same as Raa11.

(S)—((R)-1-(3-((S)-4-(allyloxy)-3-hydroxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). To a solution of 6 (2 g, 3.44 mmol) in DMF (30 ml) was added 7 (1.2g, 6.9 mmol) DIPEA (1.33 g, 0.32 mmol) and HATU (1.96 g, 5.16 mmol). Themixture was stirred at rt for 3 h before being diluted with EtOAc. Theorganic layer was washed by brine, dried over Na₂SO₄ and concentrated invacuo. The residue was purified by silica-gel column (DCM/MeOH 10:1) togive product 8 as a yellow oil (800 mg, 32%). [M+H]⁺=737.

(S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-2-hydroxy-4-oxobutanoicacid (Raa12). A solution of 8 (800 mg, 1.09 mmol) in THF (100 mL) wasadded N-Methylaniline (232 mg, 2.17 mmol) and Pd(PPh₃)₄ (115 mg, 0.1mmol). The mixture was allowed to react at room temperature under N₂atmosphere until complete conversion. The reaction mixture was chargedto silica-gel flash column directly (DCM/MeOH 10:1) to afford Raa12 (120mg, 16%) as a white solid.

FKBD Example 15 (S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoicacid (Raa13)

(S)-tert-butyl3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenylamino)-4-oxobutanoate(3). A solution of 1 (4.0 g, 14.03 mmol), 2 (7.0 g, 16.8 mmol) in DCM(150 mL) was treated with DIPEA (8 ml, 42.1 mmol) and HATU (8.0 g, 21.1mmol) at 0° C. and allowed to stir at room temperature for 15 h. Afterthis time the reaction mixture was washed with H₂O and extracted withAcOEt (50 ml*3). The organic phase was dried over Na₂SO₄ andconcentrated. The residue was purified by silica-gel flash columnchromatography (AcOEt/PE 1:10) to give compound 3 as a brown oil (9 g,90%). [M+Na]⁺=700.9

(S)-tert-butyl3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylamino)-4-oxobutanoate(4). A solution of ketone 3 (4.7 g, 6.9 mmol) in dry THF (130 mL) at−20° C. was treated with a solution of (+)-DIPChloride (27.7 mmol) inheptane (1.7 M, 16.3 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 3, then quenched with2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:10)to give compound 4 as a light yellow oil (1.7 g, 40%). [M+Na]⁺=702.8

(S)—((R)-1-(3-((S)-2-(((9H-fluoren-9yl)methoxy)carbonylamino)-4-tert-butoxy-4oxobutanamido) phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(6). A solution of 4 (1.7 g, 2.5 mmol) and 5 (1.2 g, 3.75 mmol) inCH₂Cl₂ (50 mL) was cooled to −20° C. before a solution of DCC (0.78 g,3.75 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 30 mg, 0.25 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:5) to give compound 6 as a lightyellow oil (1.0 g, 50%).

(S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoicacid (Raa13). A solution of 6 (1.0 g, 1.02 mmol) in CH₂Cl₂ (10 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (DCM/MeOH=50/1) to afford Raa13 (401 mg, 42%) as a white solid.

FKBD Example 16 (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoicacid (Raa14)

(S)-tert-butyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenylamino)-4-oxobutanoate(3). A solution of 1 (4.0 g, 14.03 mmol), 2 (7.0 g, 16.8 mmol) in DCM(150 mL) was treated with DIPEA (8 ml, 42.1 mmol) and HATU (8.0 g, 21.1mmol) at 0° C. and allowed to stir at room temperature for 15 h. Afterthis time the reaction mixture was washed with H₂O and extracted withAcOEt (50 ml*3). The organic phase was dried over Na₂SO₄ andconcentracted. The residue was purified by silica-gel flash columnchromatography (AcOEt/PE 1:10) to give compound 3 as a brown oil (9 g,90%). [M+Na]⁺=700.9

(S)-tert-butyl2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((S)-3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylamino)-4-oxobutanoate(4). A solution of ketone 3 (4.0 g, 5.9 mmol) in dry THF (80 mL) at −20°C. was treated with a solution of (+)-DIPChloride (23.6 mmol) in heptane(1.7 M, 14.0 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 3, then quenched with2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:10)to give compound 4 as a light yellow oil (2.0 g, 50%). [M+Na]⁺=702.8

(S)—((R)-1-(3-((S)-3-(((9H-fluoren-9yl)methoxy)carbonylamino)-4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(6). A solution of 4 (2.0 g, 2.9 mmol) and 5 (1.2 g, 3.82 mmol) inCH₂Cl₂ (50 mL) was cooled to −20° C. before a solution of DCC (0.91 g,4.11 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 35 mg, 0.29 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:5) to give compound 6 as a lightyellow oil (1.0 g, 50%).

(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoicacid (Raa14). A solution of 6 (1.0 g, 1.02 mmol) in CH₂Cl₂ (10 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (DCM/MeOH=50/1) to afford Raa14 (367 mg, 42%) as a white solid.

FKBD Example 17(2S,3S)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2,3-dihydroxy-4-oxobutanoicacid (Raa15)

(3R, 4S)-3,4-dihydroxydihydrofuran-2,5-dione (2). To the solution of(2R,3S)-2,3-dihydroxysuccinic acid 1 (10 g, 66.6 mmol) in DCM (100 mL)was added 2,2,2-trifluoroacetic anhydride (27.9 g, 133.2 mmol) at 25° C.The resulting solution was stirred at room temperature for 12 h. Themixture was concentrated in vacuum. The crude product was washed withpetroleum ether (100 mL) to afford 2 (6 g, 68%) as a white solid.

(2S,3S)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2,3-dihydroxy-4-oxobutanoicacid (Raa15). A mixture of 6 (2 g, 3.4 mmol), 2 (0.896 g, 6.8 mmol) andDMAP (80 mg, 0.68 mmol) in THF (60 mL) were stirred at 50° C. for 6 h.The mixture was filtered and concentrated in vacuum. The resultingresidue was purified by prep-HPLC to afford Raa15 (476 mg, 19%) as awhite solid.

FKBD Example 183-((((9H-fluoren-9-yl)methoxy)amino)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2-hydroxy-4-oxobutanoicacid (Raa16)

2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxysuccinic acid(2). To a solution of 1(10 g, 67.1 mmol) in 1,4-dioxane (150 ml) wasadded 10% NaCO₃(aq) 250 ml, and then FmocCl in 1,4-dioxane (150 ml) wasdropwisely added at 0° C. The reaction mixture was stirred at 0° C. for10 min, and then raised to rt and stirred for another 4 h. The productmixture was poured into water (500 ml), extracted with EA (200 ml) 3times. Adjusted the hydrous layer to pH=2-3 by 2M HCl, and thenextracted with DCM (200 ml) 3 times, combined the organic layer, washedwith brine (200 ml) 3 times, dried over Na₂SO₄, filtered andconcentrated to get product 2 (22 g, 88%) as white solid. [M+Na]⁺=394

2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)aceticacid (4). To a solution of 2 (5 g, 13.5 mmol) in EA (50 ml) was added 3(14 g, 135 mmol) and PTSA (0.46 g 2.7 mmol). The reaction mixture wasrefluxed for 16 h. The product mixture was concentrated directly, andthe brown residue was purified by silca gel chromatography (EA/PE=10-50%as eluent) to give 4 (3.8 g, 68.6%) as white solid. [M+Na]⁺=434

(1R)-1-(3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetamido)phenyl)-3-(3,4-dimethoxyphenyl(2S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobytanoyl)piperidine-2-carboxylate(6). To a solution of 4 (2.2 g, 5.4 mmol) in DMF (150 ml) was added HATU(3 g, 8 mmol) and DIEA (1.38 g, 10.8 mmol). 5 (2.6 g 4.5 mmol) was addedat last. The reaction mixture was stirred at rt for 1 h. Poured theproduct mixture into water (300 ml), extracted with DCM (100 ml*3),combined the organic phase and washed with brine (100 ml*5). Dried overNa₂SO₄, filtered and concentrated to get the crude. Purified by silcagel chromatography (Methanol/DCM=0-2% as eluent) to give compound 6 (3.7g, 71%) as white solid. [M+Na]⁺=996

3-((((9H-fluoren-9-yl)methoxy)amino)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2-hydroxy-4-oxobutanoicacid (Raa16). To a solution of 6 (3.7 g, 38 mmol) in THF/H₂O (10 ml/10ml) was added THF (40 ml). The reaction mixture was stirred at rt for 1h. The product mixture was evaporated directly, and the residue waspurified by silca gel chromatography (HCOOH/DCM=0-5% as eluent) to givecompound Raa16 (500 mg, 14%) as light yellow solid.

FKBD Example 192-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae1)

(E)-1-(3-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3).To the solution of 3,4,5-trimethoxybenzaldehyde 1 (5 g, 25.5 mmol) and3′-hydroxyacetophenone 2 (3.47 g, 25.5 mmol) in EtOH (50 mL) was added asolution of 10% aqueous NaOH (41 mL, 4.1 g, 101.9 mmol) at 0° C. Theresulting solution was heated to 65° C. for 2 h. The solvent wasevaporated and the residue (5.5 g, crude) was used directly for the nextstep without purification. [M+H]⁺=314.9.

3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenol (4). A solution of3 (5.5 g, crude) and 10% Pd/C (2 g) in THF (40 mL) was hydrogenated withH₂ for 4 h at room temperature. The reaction mixture was then filteredand concentrated to a solid (5.96 g, crude). [M+H—H₂O]⁺=300.9

tert-butyl2-(3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetate (5). Asolution of 4 (5.96 g, 18.8 mmol, crude) and K₂CO₃ (3.12 g, 22.6 mmol)in DMF (30 mL) was treated with tert-butyl bromoacetate (3.68 g, 18.8mmol) and allowed to stir at room temperature for 5 h. After this timethe reaction mixture was poured into ice, yellow solid was precipitated.The crude product was purified by prep-HPLC to give 5 (4.65 g, 42% (3steps)) as a yellow solid. [M+Na]⁺=454.8

tert-butyl 2-(3-(3-(3,4,5-trimethoxyphenyl)propanoyl)phenoxy)acetate(6). A solution of 5 (4.65 g, 10.75 mmol) in CH₂Cl₂ (110 mL) was treatedwith Dess-Martin periodinane (11.4 g, 26.88 mmol) and allowed to stir atroom temperature for 3 h before being quenched with a solution of 10%aqueous NaS₂O₃. The solution was extracted with CH₂Cl₂ twice. Thecombined organic layers were washed by sat. NaHCO₃, brine, dried overNa₂SO₄ and concentrated in vacuo. The crude compound was purified bysilica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6as a white solid (4.5 g, 97%). [M+Na]⁺=453.2

tert-butyl(R)-2-(3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetate(7). A solution of ketone 6 (3.98 g, 9.25 mmol) in dry THF (40 mL) at−20° C. was treated with a solution of (+)-DIPChloride (18.5 mmol) inheptane (1.7 M, 10.88 mL) at −20° C. The resulting mixture was reactedat −20° C. until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:3) togive compound 7 as a light yellow oil (2.8 g, 70%, ee >99%).[M+Na]⁺=455.2

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (1.85 g, 4.3 mmol) and 8 (2 g, 6.4 mmol) in CH₂Cl₂(15 mL) was cooled to −20° C. before a solution of DCC (1.3 g, 6.4 mmol)in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of4-(dimethylamino)pyridine (DMAP, 52.3 mg, 0.43 mmol) in CH₂Cl₂ (2 mL)under argon atmosphere. The resulting white suspension was allowed tostir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:2) to give compound 9 as a lightyellow oil (2.5 g, 80%). [M+Na]⁺=748.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae1). A solution of 9 (2.5 g, 3.44 mmol) in CH₂Cl₂ (11.5 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (11.5 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae1 (969 mg, 42%) as awhite solid.

FKBD Example 202-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae2)

(E)-1-(3-hydroxyphenyl)-3-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (3).To the solution of 2,3,4-trimethoxybenzaldehyde 1 (5 g, 25.5 mmol) and3′-hydroxyacetophenone 2 (3.47 g, 25.5 mmol) in EtOH (30 mL) was added asolution of 10% aqueous NaOH (41 mL, 4.1 g, 101.9 mmol) at 0° C. Theresulting solution was heated to 65° C. for 3 h. The solvent wasevaporated and the residue was purified by silica-gel flash columnchromatography (AcOEt/PE 1:5) to give compound 3 as a yellow oil (6.6 g,83%). [M+H]⁺=314.9

3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenol (4). A solution of3 (6.6 g, 21 mmol) and 10% Pd/C (3 g) in THF (30 mL) was hydrogenatedwith H₂ for 16 h at room temperature. The reaction mixture was thenfiltered and concentrated to a colorless oil (8 g, crude).[M+H—H₂O]⁺=301.0

tert-butyl2-(3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetate (5). Asolution of 4 (8 g, 25 mmol, crude) and K₂CO₃ (4.19 g, 30 mmol) in DMF(30 mL) was treated with tert-butyl bromoacetate (5.92 g, 30 mmol) andallowed to stir at room temperature for 6 h. After this time thereaction mixture was quenched by H₂O and extracted with EtOAc twice. Thecombined organic layers were washed by brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 6 as acolorless oil (8.2 g, 90% (2 steps)). [M+H—H₂O—tBu]⁺=358.8

tert-butyl 2-(3-(3-(2,3,4-trimethoxyphenyl)propanoyl)phenoxy)acetate(6). A solution of 5 (5.75 g, 13.29 mmol) in CH₂Cl₂ (30 mL) was treatedwith Dess-Martin periodinane (11.28 g, 26.59 mmol) and allowed to stirat room temperature for 2 h before being quenched with a solution of 10%aqueous NaS₂O₃. The solution was extracted with CH₂Cl₂ twice. Thecombined organic layers were washed by sat. NaHCO₃, brine, dried overNa₂SO₄ and concentrated in vacuo. The crude compound was purified bysilica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6as a yellow oil (5 g, 87%).

tert-butyl(R)-2-(3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetate(7). A solution of ketone 6 (5 g, 11.61 mmol) in dry THF (50 mL) at −20°C. was treated with a solution of (+)-DIPChloride (23.23 mmol) inheptane (1.7 M, 13.66 mL) at −20° C. The resulting mixture was reactedat −20° C. until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (3.4 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:3) togive compound 7 as a light yellow oil (4 g, 80%, ee 83%).[M+H—H₂O−tBu]⁺=358.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (2 g, 4.62 mmol) and 8 (2.16 g, 6.93 mmol) inCH₂Cl₂ (23 mL) was cooled to −20° C. before a solution of DCC (1.43 g,6.93 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 57 mg, 0.46 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:2) to give compound 9 as a lightyellow oil (2.13 g, 64%). [M+Na]⁺=748.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae2). A solution of 9 (2.13 g, 2.93 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae2 (508 mg, 25%) as a paleyellow solid.

FKBD Example 212-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae3)

(E)-1-(3-hydroxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (3).To the solution of 2,4,5-trimethoxybenzaldehyde 1 (4.5 g, 22.96 mmol)and 3′-hydroxyacetophenone 2 (3.1 g, 22.96 mmol) in EtOH (50 mL) wasadded a solution of 10% aqueous KOH (15 mL, 5.1 g, 91.84 mmol) at 0° C.The resulting solution was heated to 60° C. for 4 h. The solvent wasevaporated and the residue was purified by silica-gel flash columnchromatography (AcOEt/PE 1:3) to give compound 3 as a yellow oil (5.9 g,82%). [M+H]⁺=315.1

tert-butyl (E)-2-(3-(3-(2,4,5-trimethoxyphenyl)acryloyl)phenoxy)acetate(4). A solution of 3 (7.4 g, 23.6 mmol) and K₂CO₃ (3.9 g, 28.3 mmol) inDMF (200 mL) was treated with tert-butyl bromoacetate (5.5 g, 28.3 mmol)and allowed to stir at room temperature for 4 h. After this time thereaction mixture was quenched by H₂O and extracted with EtOAc twice. Thecombined organic layers were washed by brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 4 as acolorless oil (8 g, 80%). [M+H]⁺=429.3

tert-butyl 2-(3-(3-(2,4,5-trimethoxyphenyl)propanoyl)phenoxy)acetate(5). A solution of 4 (8 g, 18.69 mmol) and 10% Pd/C (1 g) in THF (200mL) was hydrogenated with H₂ for 8 h at room temperature. The reactionmixture was then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 5as a colorless oil (6 g, 75%). [M+Na]⁺=453.2

tert-butyl(R)-2-(3-(1-hydroxy-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)acetate(6). A solution of ketone 5 (6 g, 13.95 mmol) in dry THF (60 mL) at −20°C. was treated with a solution of (+)-DIPChloride (41.86 mmol) inheptane (1.7 M, 24.6 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 5, then quenched with2,2′-(ethylenedioxy)diethylamine (5.9 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:1) togive compound 6 as a light yellow oil (5.5 g, 92%, ee >99%).).[M+Na]⁺=455.2

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,4,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (1.85 g, 4.28 mmol) and 7 (2 g, 6.42 mmol) inCH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (1.33 g,6.42 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 52 mg, 0.43 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:1) to give compound 8 as a lightyellow oil (2.35 g, 76%). [M+Na]⁺=747.9

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae3). A solution of 8 (2.35 g, 3.24 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae3 (815 mg, 37%) as a paleyellow solid.

FKBD Example 222-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae4)

(E)-1-(3-hydroxyphenyl)-3-(2,3,5-trimethoxyphenyl)prop-2-en-1-one (3).To the solution of 2,3,5-trimethoxybenzaldehyde 1 (6 g, 30.6 mmol) and1-(3-hydroxyphenyl)ethan-1-one 2 (4.2 g, 30.6 mmol) in EtOH (50 mL) wasadded a solution of 10% aqueous NaOH (50 mL, 122.4 mmol) at 0° C. Theresulting solution was stirred at room temperature for 12 h. Thesolution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C.,generated a large of yellow solid. Then the mixture was fitered and thesolid was washed with water (50 mL) to afford 3 (5.5 g, 57%) as a yellowsolid. [M+H]⁺=315.2.

tert-butyl (E)-2-(3-(3-(2,3,5-trimethoxyphenyl)acryloyl)phenoxy) acetate(4). A solution of 3 (5.5 g, 17.4 mmol) and K₂CO₃ (4.82 g, 34.9 mmol) inDMF (40 mL) was treated with tert-butyl bromoacetate (4.06 g, 20.9 mmol)and allowed to stir at room temperature for 12 h. After this time thereaction mixture was poured into ice, yellow solid was precipitated. Themixture was filtered and the solid was washed with water (30 mL). Thecrude product was washed with petroleum ether (50 mL) to give 4 (7 g,93%) as a yellow solid. [M+H]⁺=428.8

tert-butyl 2-(3-(3-(2,3,5-trimethoxyphenyl)propanoyl)phenoxy)acetate(5). A solution of 4 (7 g, 11.68 mmol) and 10% Pd/C (1 g) in THF (100mL) was hydrogenated with H₂ for 4 h at room temperature. The reactionmixture was then filtered and concentrated. The crude product waspurified by column chromatography on silica gel to give 5 (3.5 g, 50%)as a yellow oil. [M+Na]⁺=452.9.

tert-butyl(R)-2-(3-(1-hydroxy-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)acetate(6). A solution of ketone 5 (3.5 g, 8.14 mmol) in dry THF (30 mL) at−20° C. was treated with a solution of (+)-DIPChloride (16.2 mmol) inheptane (1.7 M, 9.5 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (2.4 g) by forming an insolublecomplex. After stirring at room temperature for another 30 min, thesuspension was filtered through a pad of celite and concentrated. Thecrude compound was purified by silica-gel flash column chromatography(AcOEt/PE 1:4) to give compound 6 (2.2 g, 63%, ee 97% vs racemate) as alight yellow oil. [M+Na]⁺=454.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,3,5-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (2.2 g, 5.09 mmol) and 8 (1.89 g, 6.1 mmol) inCH₂Cl₂ (15 mL) was cooled to −20° C. before a solution of DCC (1.36 g,6.6 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (62 mg, 0.5 mmol) in CH₂Cl₂ (2 mL)under argon atmosphere. The resulting white suspension was allowed tostir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:2) to give compound 8 (1.8 g, 49%) asa light yellow oil. [M+Na]⁺=748.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae4). A solution of 8 (1.8 g, 2.48 mmol) in CH₂Cl₂ (10 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae4 (652 mg, 39%) as afaint yellow solid.

FKBD Example 232-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae5)

(E)-1-(3-hydroxyphenyl)-3-(2,3,6-trimethoxyphenyl)prop-2-en-1-one (3).To the solution of 2,3,6-trimethoxybenzaldehyde 1 (5 g, 25.48 mmol) and3′-hydroxyacetophenone 2 (3.47 g, 25.48 mmol) in EtOH (40 mL) was addeda solution of 40% aqueous KOH (15 mL, 5.7 g, 101.92 mmol) at 0° C. Theresulting solution was reacted at room temperature for 4 h. The solventwas evaporated and the residue was purified by silica-gel flash columnchromatography (AcOEt/PE 1:2) to give compound 3 as a yellow oil (4 g,50%). [M+H]⁺=315.2

tert-butyl (E)-2-(3-(3-(2,3,6-trimethoxyphenyl)acryloyl)phenoxy)acetate(4). A solution of 3 (3.5 g, 11.15 mmol) and K₂CO₃ (1.85 g, 13.37 mmol)in DMF (60 mL) was treated with tert-butyl bromoacetate (2.6 g, 13.37mmol) and allowed to stir at room temperature for 4 h. After this timethe reaction mixture was quenched by H₂O and extracted with EtOAc twice.The combined organic layers were washed by brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:4) to give compound 4 as ayellow oil (4.7 g, 98%). [M+H]⁺=429.0

tert-butyl 2-(3-(3-(2,3,6-trimethoxyphenyl)propanoyl)phenoxy)acetate(5). A solution of 4 (4.6 g, 10.75 mmol) and 10% Pd/C (0.5 g) in THF (70mL) was hydrogenated with H₂ for 4 h at room temperature. The reactionmixture was then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 5as a colorless oil (2.9 g, 63%). [M+Na]⁺=453.3

tert-butyl(R)-2-(3-(1-hydroxy-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)acetate(6). A solution of ketone 5 (2.9 g, 6.7 mmol) in dry THF (30 mL) at −20°C. was treated with a solution of (+)-DIPChloride (13.48 mmol) inheptane (1.7 M, 7.9 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 5, then quenched with2,2′-(ethylenedioxy)diethylamine (1.96 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:5) togive compound 6 as a light yellow oil (2.4 g, 83%, ee >99%).[M+Na]⁺=454.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,3,6-trimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine2-carboxylate (8). A solution of 6 (1.46 g, 3.45 mmol) and 7 (1.6 g,5.17 mmol) in CH₂Cl₂ (18 mL) was cooled to −20° C. before a solution ofDCC (1.065 g, 5.17 mmol) in CH₂Cl₂ (5 mL) was added, followed by theaddition of a solution of 4-(dimethylamino)pyridine (DMAP, 43 mg, 0.35mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting whitesuspension was allowed to stir at −20° C. for 2 h. The reaction mixturewas then filtered, evaporated, and the crude compound was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 8as a light yellow oil (1.7 g, 68%).

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)aceticacid (Rae5). A solution of 8 (1.7 g, 2.34 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae5 (494 mg, 31%) as a paleyellow solid.

FKBD Example 242-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae9)

2-(tert-butoxy)-3,4-dimethoxybenzaldehyde (2). To a solution of2-hydroxy-3,4-dimethoxybenzaldehyde 1 (2.77 g, 15.2 mmol) in anhydroustoluene (30 mL) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine 2(29.1 mL, 122 mmol) under Ar. atmosphere. The mixture was stirred at 80°C. for 6 h, then the solvent was evaporated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 2as a yellow solid (2.965 g, 82%). [M+Na]⁺=261.1

(E)-3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4). To thesolution of 2 (2.965 g, 12.4 mmol) and 3′-hydroxyacetophenone 3 (2.03 g,14.9 mmol) in EtOH (50 mL) was added a solution of 40% aqueous KOH (6.98g, 49.8 mmol) at 0° C. The resulting solution was stirred at 60° C. for4 h. The solution was poured into water and acidified to pH 4 with a 1 MHCl aqueous solution, extracted with EtOAc twice. The combined organiclayers were washed by brine, dried over Na₂SO₄ and concentrated invacuo. The crude compound was purified by silica-gel flash columnchromatography (AcOEt/PE 1:1) to give compound 4 as a yellow oil (3.2 g,72%). [M+Na]⁺=378.9

tert-butyl(E)-2-(3-(3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)acryloyl)phenoxy)acetate(5). A solution of 4 (3.2 g, 9 mmol) and K₂CO₃ (1.49 g, 10.8 mmol) inDMF (30 mL) was treated with tert-butyl bromoacetate (1.58 mL, 10.8mmol) and allowed to stir at room temperature for 5 h. After this timethe reaction mixture was quenched by H₂O and extracted with EtOAc twice.The combined organic layers were washed by brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:4) to give compound 5 as ayellow oil (4 g, 95%).[M+Na]⁺=493.3

tert-butyl2-(3-(3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate(6). A solution of 5 (4 g, 8.5 mmol) and 10% Pd/C (0.8 g) in THF (50 mL)was hydrogenated with H₂ for 3 h at room temperature. The reactionmixture was then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6as a colorless oil (2.878 g, 72%). [M+Na]⁺=495.3

tert-butyl(R)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(7). A solution of ketone 6 (2.878 g, 6.1 mmol) in dry THF (30 mL) at−20° C. was treated with a solution of (+)-DIPChloride (24.4 mmol) inheptane (1.7 M, 14.3 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (3.6 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:3) togive compound 7 as a light yellow oil (2 g, 70%, ee >99%). [M+Na]⁺=497.0

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (1.8 g, 3.793 mmol) and 8 (1.77 g, 5.69 mmol) inCH₂Cl₂ (13 mL) was cooled to −20° C. before a solution of DCC (1.17 g,5.69 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 46 mg, 0.379 mmol) inCH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 9 as a lightyellow oil (2.5 g, 86%). [M+Na]⁺=790.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae9): A solution of 9 (2.5 g, 3.26 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae9 (636 mg, 30%) as a paleyellow solid.

FKBD Example 252-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae10)

3-(tert-butoxy)-4,5-dimethoxybenzaldehyde (2). To a solution of3-hydroxy-4,5-dimethoxybenzaldehyde 1 (2.77 g, 15.2 mmol) in anhydroustoluene (30 mL) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine 2(29.1 mL, 122 mmol) under Ar. atmosphere. The mixture was stirred at 80°C. for 6 h, then the solvent was evaporated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 2as a yellow solid (3.126 g, 86%). [M+H]⁺=239.0

(E)-3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one(4). To the solution of 2 (3.126 g, 13 mmol) and 3′-hydroxyacetophenone3 (2.14 g, 15.7 mmol) in EtOH (30 mL) was added a solution of 40%aqueous KOH (7.36 g, 52 mmol) at 0° C. The resulting solution wasstirred at 60° C. for 4 h. The solution was poured into water andacidified to pH 4 with a 1 M HCl aqueous solution, extracted with EtOActwice. The combined organic layers were washed by brine, dried overNa₂SO₄ and concentrated in vacuo. The crude compound was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 4as a yellow oil (2.489 g, 54%). [M+H]⁺=357.0

tert-butyl(E)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)acryloyl)phenoxy)acetate(5). A solution of 4 (2.489 g, 6.98 mmol) and K₂CO₃ (1.16 g, 8.38 mmol)in DMF (30 mL) was treated with tert-butyl bromoacetate (1.2 mL, 8.38mmol) and allowed to stir at room temperature for 5 h. After this timethe reaction mixture was quenched by H₂O and extracted with EtOAc twice.The combined organic layers were washed by brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:4) to give compound 5 as ayellow oil (3.1 g, 95%). [M+H]⁺=471.0

tert-butyl2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate(6). A solution of 5 (3.1 g, 6.59 mmol) and 10% Pd/C (0.5 g) in THF (50mL) was hydrogenated with H₂ for 3 h at room temperature. The reactionmixture was then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6as a colorless oil (2.88 g, 93%). [M+Na]⁺=495.3

tert-butyl(R)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(7). A solution of ketone 6 (2.868 g, 6.07 mmol) in dry THF (30 mL) at−20° C. was treated with a solution of (+)-DIPChloride (12.1 mmol) inheptane (1.7 M, 7.1 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (3.6 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:3) togive compound 7 as a light yellow oil (2.03 g, 70%, ee >99% vsracemate). [M+Na]⁺=497.3

(R)-1-(3-(3-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (2.03 g, 4.3 mmol) and 8 (2 g, 6.4 mmol) in CH₂Cl₂(43 mL) was cooled to −20° C. before a solution of DCC (1.3 g, 6.4 mmol)in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of4-(dimethylamino)pyridine (DMAP, 52.3 mg, 0.43 mmol) in CH₂Cl₂ (2 mL)under argon atmosphere. The resulting white suspension was allowed tostir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:3) to give compound 9 as a lightyellow oil (2.5 g, 76%). [M+Na]⁺=790.3

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae10). A solution of 9 (2.5 g, 3.26 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae10 (1.334 g, 62%) as apale yellow solid.

FKBD Example 262-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae11)

2-(tert-butoxy)-4,5-dimethoxybenzaldehyde (2). To a solution of2-hydroxy-4,5-dimethoxybenzaldehyde 1 (3 g, 16.5 mmol) in anhydroustoluene (15 mL) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine 2(31.6 mL, 132 mmol) under Ar. atmosphere. The mixture was stirred at 80°C. for 6 h, then the solvent was evaporated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 2as a yellow solid (3.875 g, 99%). [M+Na]⁺=261.2

(E)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one(4). To the solution of 2 (3.875 g, 16.3 mmol) and3′-hydroxyacetophenone 3 (2.436 g, 17.9 mmol) in EtOH (50 mL) was addeda solution of 40% aqueous KOH (8.5 mL, 3.65 g, 65.2 mmol) at 0° C. Theresulting solution was stirred at 60° C. for 4 h. The solution waspoured into water and acidified to pH 4 with a 1M HCl aqueous solution,extracted with EtOAc twice. The combined organic layers were washed bybrine, dried over Na₂SO₄ and concentrated in vacuo. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:1) togive compound 4 as a yellow oil (5.2 g, 90%). [M+H]⁺=357.2

tert-butyl(E)-2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)acryloyl)phenoxy)acetate(5). A solution of 4 (5.2 g, 14.59 mmol) and K₂CO₃ (2.4 g, 17.5 mmol) inDMF (50 mL) was treated with tert-butyl bromoacetate (2.55 mL, 17.5mmol) and allowed to stir at room temperature for 5 h. After this timethe reaction mixture was quenched by H₂O and extracted with EtOAc twice.The combined organic layers were washed by brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:4) to give compound 5 as ayellow oil (6 g, 88%). [M+H]⁺=471.0

tert-butyl2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate(6). A solution of 5 (6 g, 12.75 mmol) and 10% Pd/C (1 g) in THF (70 mL)was hydrogenated with H₂ for 4 h at room temperature. The reactionmixture was then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6as a colorless oil (4.5 g, 75%). [M+Na]⁺=495.3

tert-butyl(R)-2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(7). A solution of ketone 6 (4.5 g, 9.5 mmol) in dry THF (45 mL) at −20°C. was treated with a solution of (+)-DIPChloride (19 mmol) in heptane(1.7 M, 11.2 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:3) togive compound 7 as a light yellow oil (3.2 g, 70%, ee >99% vs racemate).[M+Na]⁺=496.7

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (1.93 g, 4.07 mmol) and 8 (1.9 g, 6.103 mmol) inCH₂Cl₂ (43 mL) was cooled to −20° C. before a solution of DCC (1.26 g,6.103 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 50 mg, 0.407 mmol) inCH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 9 as a lightyellow oil (2.1 g, 67%). [M+Na]⁺=790.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae11). A solution of 9 (2.1 g, 2.73 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae11 (638 mg, 23%) as apale yellow solid.

FKBD Example 272-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae12)

(E)-3-(3-fluoro-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one(3). To the solution of 3-fluoro-4,5-dimethoxybenzaldehyde 1 (4.5 g,24.4 mmol) and 1-(3-hydroxyphenyl)ethan-1-one 2 (3.3 g, 24.4 mmol) inEtOH (60 mL) was added a solution of 10% aqueous NaOH (40 mL, 97.6 mmol)at 0° C. The resulting solution was stirred at room temperature for 12h. The solution was adjusted to pH 4 by added 4M aqueous HCl dropwise at0° C., generated a large of yellow solid. Then the mixture was fiteredand the solid was washed with water (50 mL) to afford 3 (4 g, 54%) as ayellow solid. [M+H]⁺=303.1

tert-butyl(E)-2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)acryloyl)phenoxy)acetate (4).A solution of 3 (4 g, 13.2 mmol) and K₂CO₃ (3.65 g, 26.4 mmol) in DMF(30 mL) was treated with tert-butyl bromoacetate (3.08 g, 15.8 mmol) andallowed to stir at room temperature for 5 h. After this time thereaction mixture was poured into ice, yellow solid was precipitated. Themixture was filtered and the solid was washed with water (30 mL). Thecrude product was purified by column chromatography on silica gel(AcOEt/PE 1:4) to give 4 (5.2 g, 94%) as a yellow solid. [M+Na]⁺=438.7

tert-butyl2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(5). A solution of 4 (5.2 g, 12.5 mmol) and 10% Pd/C (1 g) in THF (100mL) was hydrogenated with H₂ for 2 h at room temperature. The reactionmixture was then filtered and concentrated. The crude product waspurified by column chromatography on silica gel to give 5 (5 g, 96%) asa yellow oil. [M+Na]⁺=443.2

tert-butyl2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate (6). Asolution of 5 (5 g, 11.9 mmol) in CH₂Cl₂ (100 mL) was treated withDess-Martin periodinane (15.2 g, 36 mmol) and allowed to stir at roomtemperature for 2 h before being quenched with a solution of 10% aqueousNaS2O₃. The solution was extracted with CH₂Cl₂ twice. The combinedorganic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 6 as a whitesolid (4 g, 80%). [M+Na]⁺=441.2

tert-butyl(R)-2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(7). A solution of ketone 6 (4 g, 9.56 mmol) in dry THF (30 mL) at −20°C. was treated with a solution of (+)-DIPChloride (19.1 mmol) in heptane(1.7 M, 11.2 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (2.8 g) by forming an insolublecomplex. After stirring at room temperature for another 30 min, thesuspension was filtered through a pad of celite and concentrated. Thecrude compound was purified by silica-gel flash column chromatography(AcOEt/PE 1:4) to give compound 6 (2.2 g, 55%, ee >99%) as a lightyellow oil. [M+Na]⁺=442.7

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (2.2 g, 5.23 mmol) and 8 (1.80 g, 5.76 mmol) inCH₂Cl₂ (20 mL) was cooled to −20° C. before a solution of DCC (1.4 g,6.79 mmol) in CH₂Cl₂ (10 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (63 mg, 0.52 mmol) in CH₂Cl₂ (2mL) under argon atmosphere. The resulting white suspension was allowedto stir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:3) to give compound 9 (1.8 g, 48%) asa light yellow oil. [M+Na]⁺=736.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae12). A solution of 9 (1.8 g, 2.52 mmol) in CH₂Cl₂ (10 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae12 (590 mg, 35%) as afaint yellow solid.

FKBD Example 282-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae13)

(E)-3-(2-fluoro-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one(3). To the solution of 2-fluoro-4,5-dimethoxybenzaldehyde 1 (4.5 g,24.4 mmol) and 1-(3-hydroxyphenyl)ethan-1-one 2 (3.3 g, 24.4 mmol) inEtOH (60 mL) was added a solution of 10% aqueous NaOH (40 mL, 97.6 mmol)at 0° C. The resulting solution was stirred at 65° C. for 6 h. Thesolution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C.,generated a large of yellow solid. Then the mixture was fitered and thesolid was washed with water (50 mL) to afford 3 (7 g, 94%) as a yellowsolid. [M+H]⁺=302.8

3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenol (4). Asolution of 3 (7 g, 23.1 mmol) and 10% Pd/C (2 g) in THF (150 mL) washydrogenated with H₂ for 12 h at room temperature. The reaction mixturewas then filtered and concentrated. The crude product was purified bycolumn chromatography on silica gel to give 4 (7 g, 98%) as a yellowoil. [M+Na]⁺=328.8

tert-butyl2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(5). A solution of 4 (7 g, 23.1 mmol) and K₂CO₃ (7 g, 50.6 mmol) in DMF(200 mL) was treated with tert-butyl bromoacetate (6.7 g, 34.5 mmol) andallowed to stir at room temperature for 24 h. After this time thereaction mixture was poured into ice, yellow solid was precipitated. Themixture was filtered and the solid was washed with water (300 mL). Thecrude product was purified by column chromatography on silica gel(AcOEt/PE 1:6) to give 5 (8 g, 82%) as a yellow solid. [M+Na]⁺=443.2

tert-butyl2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate (6). Asolution of 5 (8 g, 19 mmol) in CH₂Cl₂ (100 mL) was treated withDess-Martin periodinane (16 g, 38 mmol) and allowed to stir at roomtemperature for 2 h before being quenched with a solution of 10% aqueousNaS2O₃. The solution was extracted with CH₂Cl₂ twice. The combinedorganic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 6 as ayellow solid (6.3 g, 78%). [M+Na]⁺=440.7

tert-butyl(R)-2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(7). A solution of ketone 6 (6.3 g, 15.07 mmol) in dry THF (60 mL) at−20° C. was treated with a solution of (+)-DIPChloride (45.2 mmol) inheptane (1.7 M, 26.5 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (6.6 g) by forming an insolublecomplex. After stirring at room temperature for another 30 min, thesuspension was filtered through a pad of celite and concentrated. Thecrude compound was purified by silica-gel flash column chromatography(AcOEt/PE 1:3) to give compound 7 (4.3 g, 68%, ee >99%) as a lightyellow oil. [M+Na]⁺=443.2

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(9). A solution of 7 (1.6 g, 3.81 mmol) and 8 (1.77 g, 5.71 mmol) inCH₂Cl₂ (20 mL) was cooled to −20° C. before a solution of DCC (1.17 g,5.71 mmol) in CH₂Cl₂ (10 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (50 mg, 0.38 mmol) in CH₂Cl₂ (2mL) under argon atmosphere. The resulting white suspension was allowedto stir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:2) to give compound 9 (1.7 g, 62%) asa light yellow oil. [M+Na]⁺=736.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae13). A solution of 9 (1.7 g, 2.38 mmol) in CH₂Cl₂ (10 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae13 (520 mg, 33%) as afaint yellow solid.

FKBD Example 292-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae14)

(E)-3-(2-fluoro-3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one(3). To the solution of 1(5.0 g, 27.17 mmol) and 2 (4.10 g, 29.89 mmol)in EtOH (150 mL) was added a solution of 40% aqueous KOH (15.22 g,108.70 mmol) at 0° C. The resulting solution was heated to 35° C. for 2h. The solvent was evaporated and the residue (4.8 g 58%) was useddirectly for the next step without purification. [M+H]⁺=303.0

(E)-tert-butyl2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)acryloyl)phenoxy)acetate (4). Asolution of 3 (5.0 g, 16.55 mmol, crude) and K₂CO₃ (2.74 g, 19.87 mmol)in DMF (40 mL) was treated with tert-butyl bromoacetate (3.9 g, 19.87mmol) and allowed to stir at room temperature for 5 h. After this timethe reaction mixture was poured into ice, yellow solid was precipitated.The mixture was filtered and the solid was washed with water (30 mL).The crude product was purified by column chromatography on silica gel togive 4 (6.0 g, 80%) as a yellow solid. [M+Na]⁺=439.2

tert-butyl2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate (5). Asolution of 4 (4.0 g, 9.62 mmol) and 10% Pd/C (1.0 g) in THF (150 mL)was hydrogenated with H₂ for 4 h at room temperature. The reactionmixture was then filtered and concentrated. The crude product waspurified by column chromatography on silica gel to give 5 (2.8 g, 70%)as a yellow oil. [M+Na]⁺=440.8

tert-butyl(R)-2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(6). A solution of ketone 5 (2.8 g, 6.7 mmol) in dry THF (30 mL) at −20°C. was treated with a solution of (+)-DIPChloride (26.8 mmol) in heptane(1.7 M, 15.7 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (3.96 g) by forming an insolublecomplex. After stirring at room temperature for another 30 min, thesuspension was filtered through a pad of celite and concentrated. Thecrude compound was purified by silica-gel flash column chromatography(AcOEt/PE 1:4) to give compound 6 (1.3 g, 46%, ee >99%) as a lightyellow oil. [M+Na]⁺=442.7

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (1.3 g, 3.09 mmol) and 7 (1.25 g, 4.02 mmol) inCH₂Cl₂ (15 mL) was cooled to −20° C. before a solution of DCC (0.83 g,4.02 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (40 mg, 0.31 mmol) in CH₂Cl₂ (2mL) under argon atmosphere. The resulting white suspension was allowedto stir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:2) to give compound 8 (1.4 g, 63%) asa light yellow oil. [M+Na]⁺=736.3

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae14). A solution of 8 (1.4 g, 1.96 mmol) in CH₂Cl₂ (10 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae14 (585 mg, 45%) as afaint yellow solid.

FKBD Example 302-(3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae16)

(E)-3-(3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3). Tothe solution of 3,4-dimethoxybenzaldehyde 1 (17.6 g, 105.8 mmol) and3′-hydroxyacetophenone 2 (12 g, 88.2 mmol) in EtOH (160 mL) was added asolution of 40% aqueous KOH (44 mL, 20 g, 352.8 mmol) at 0° C. Theresulting solution was stirred at rt for 2 h, before being poured intoice-H₂₀, the solution was acidified with 1M HCl solution and extractedwith EtOAc. The combined organic layers were dried over Na₂SO₄ andconcentrated in vacuo. The residue was recrystallized from EtOAc-PE togive the pale yellow powder (23 g, 92%). [M+H]⁺=285.2

3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (4). A solution of 3(16 g, 56.3 mmol) and 10% Pd/C (1.6 g) in THF (150 mL) was hydrogenatedwith H₂ for 4 h at room temperature. The reaction mixture was thenfiltered and concentrated to a solid (16.3 g, quant.). [M+Na]⁺=311.2

tert-butyl 2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(5). A solution of 4 (16.3 g, 56.53 mmol) and K₂CO₃ (9.4 g, 67.83 mmol)in DMF (150 mL) was treated with tert-butyl bromoacetate (9.9 mL, 67.83mmol) and allowed to stir at room temperature for 5 h. After this timethe reaction mixture was poured into ice, yellow solid was precipitated(20 g, 88%). [M+Na]⁺=424.9.

tert-butyl 2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate (6). Asolution of 5 (20 g, 49.7 mmol) in CH₂Cl₂ (400 mL) was treated withDess-Martin periodinane (63 g, 149 mmol) and allowed to stir at roomtemperature for 3 h before being quenched with a solution of 10% aqueousNaS2O₃. The solution was extracted with CH₂Cl₂ twice. The combinedorganic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ andconcentrated in vacuo. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 6 as a whitesolid (11 g, 55%). [M+Na]⁺=423.3.

tert-butyl(R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (7). Asolution of ketone 6 (11.156 g, 27.9 mmol) in dry THF (100 mL) at −20°C. was treated with a solution of (+)-DIPChloride (83.6 mmol) in heptane(1.7 M, 49 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (11.5 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:3) togive compound 7 as a light yellow oil (6.3 g, 58%, ee >99%).[M+Na]⁺=425.3.

1-((9H-fluoren-9-yl)methyl)3-((R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl)(S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-1,3-dicarboxylate(9). A solution of 7 (1.224 g, 3 mmol) and 8 (2.44 g, 4.56 mmol) inCH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (0.94 g,4.56 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 37 mg, 0.3 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:2) to give compound 9 as a lightyellow oil (1.8 g, 70%). [M+Na]⁺=940.7.

2-(3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae16). A solution of 9 (1.8 g, 1.96 mmol) in CH₂Cl₂ (11.5 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (11.5 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae16 (964 mg, 57%) as awhite solid.

FKBD Example 312-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae17)

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carboxylate(2). To the solution of 1(1.0 g, 1.09 mmol) in DMF (5 mL) was added TBAF(2.5 ml, 1.0 M, 2.55 mmol) at 0° C. The resulting solution was warmed toroom temperature for 5 h. After this time the reaction mixture wasdiluted with DCM and washed with sat. NaHCO₃ aqueous solution and brine.The organic layer was concentrated in vacuo, the residue was purified bysilica-gel flash column chromatography (DCM/MeOH 50:1) to give compound2 as a colorless oil (670 mg, 80%). [M+H]⁺=696.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carboxylate(3). A solution of 2 (670 mg, 0.96 mmol) in CHOOH (1.5 mL) was treatedwith an aqueous solution of formaldehyde (37% in water,0.77 ml, 1.15mmol) and allowed to stir at 50° C. for 1 h. After this time thereaction mixture was purified with DCM/MeOH/AcOH=100/1/0.5% to give 3(500 mg, 73%) as a colorless oil. [M+H]⁺=710.9

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)aceticacid (Rae17). A solution of 9 (0.5 g, 0.7 mmol) in HCOOH (40 mL) washeated to 40° C. for 2 h. The reaction mixture was charged to silica-gelflash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae17 (368.7mg, 80%) as a white solid.

FKBD Example 322-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-4-fluorophenoxy)aceticacid (Rae18)

(E)-3-(3,4-dimethoxyphenyl)-1-(2-fluoro-5-hydroxyphenyl)prop-2-en-1-one(3). To the solution of 3,4-dimethoxybenzaldehyde 1 (5 g, 30.1 mmol) and1-(2-fluoro-5-hydroxyphenyl)ethan-1-one 2 (4.6 g, 30.1 mmol) in EtOH (60mL) was added a solution of 10% aqueous NaOH (50 mL, 120.4 mmol) at 0°C. The resulting solution was stirred at room temperature for 12 h. Thesolution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C.,generated a large of yellow solid. Then the mixture was fitered and thesolid was washed with water (50 mL) to afford 3 (9 g, 99%) as a yellowsolid. [M+H]⁺=303.2

3-(3,4-dimethoxyphenyl)-1-(2-fluoro-5-hydroxyphenyl)propan-1-one (4). Asolution of 3 (9 g, 29.8 mmol) and 10% Pd/C (2 g) in THF (200 mL) washydrogenated with H₂ for 12 h at room temperature. The reaction mixturewas then filtered and concentrated. The crude product was used to thenext step without any further purification. [M+H]⁺=304.8

tert-butyl2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)-4-fluorophenoxy)acetate (5). Asolution of 4 (10 g, 32.8 mmol) and K₂CO₃ (9 g, 65.6 mmol) in DMF (200mL) was treated with tert-butyl bromoacetate (7.7 g, 39.3 mmol) andallowed to stir at room temperature for 8 h. After this time thereaction mixture was poured into ice, yellow solid was precipitated. Themixture was filtered and the solid was washed with water (300 mL). Thecrude product was purified by column chromatography on silica gel(AcOEt/PE 1:6) to give 5 (4 g, 32%, 2 steps) as a yellow oil.[M+Na]⁺=441.0

tert-butyl(R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-4-fluorophenoxy)acetate(6). A solution of ketone 5 (4 g, 9.56 mmol) in dry THF (30 mL) at −20°C. was treated with a solution of (+)-DIPChloride (28.68 mmol) inheptane (1.7 M, 16.8 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 6, then quenched with2,2′-(ethylenedioxy)diethylamine (4.2 g) by forming an insolublecomplex. After stirring at room temperature for another 30 min, thesuspension was filtered through a pad of celite and concentrated. Thecrude compound was purified by silica-gel flash column chromatography(AcOEt/PE 1:3) to give compound 6 (2 g, 50%, ee 93%) as a light yellowoil. [M+Na]⁺=442.7

(R)-1-(5-(2-(tert-butoxy)-2-oxoethoxy)-2-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (2 g, 4.76 mmol) and 7 (2.22 g, 7.14 mmol) inCH₂Cl₂ (20 mL) was cooled to −20° C. before a solution of DCC (1.47 g,7.14 mmol) in CH₂Cl₂ (10 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (60 mg, 0.47 mmol) in CH₂Cl₂ (2mL) under argon atmosphere. The resulting white suspension was allowedto stir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:2) to give compound 8 (1.8 g, 45%) asa light yellow oil. [M+Na]⁺=736.3

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-4-fluorophenoxy)aceticacid (Rae18). A solution of 8 (1.7 g, 2.52 mmol) in CH₂Cl₂ (10 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae18 (705 mg, 42%) as awhite solid.

FKBD Example 332-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-5-fluorophenoxy)aceticacid (Rae-19)

(E)-3-(3,4-dimethoxyphenyl)-1-(3-fluoro-5-hydroxyphenyl)prop-2-en-1-one(3). To the solution of 3,4-dimethoxybenzaldehyde 1 (6.391 g, 38.5 mmol)and 1-(3-fluoro-5-hydroxyphenyl)ethan-1-one 2 (5.39 g, 35 mmol) in EtOH(70 mL) was added a solution of 40% aqueous KOH (19.6 g, 140 mmol) at 0°C. The resulting solution was reacted at room temperature for 4 h. Theyellow solid was filtrated to give compound 3 (8.3 g, 78%). [M+H]⁺=303.0

tert-butyl(E)-2-(3-(3-(3,4-dimethoxyphenyl)acryloyl)-5-fluorophenoxy)acetate (4).A solution of 3 (8.3 g, 27.5 mmol) and K₂CO₃ (4.55 g, 32.9 mmol) in DMF(80 mL) was treated with tert-butyl bromoacetate (6.4 g, 32.9 mmol) andallowed to stir at room temperature for 4 h. After this time thereaction mixture was quenched by H₂O and extracted with EtOAc twice. Thecombined organic layers were concentrated in vacuo, which was used forthe next step without purification (11.11 g, 97%). [M+Na]⁺=439.2

tert-butyl2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)-5-fluorophenoxy)acetate (5). Asolution of 4 (11.11 g, 26.7 mmol) and 10% Pd/C (1.11 g) in THF (200 mL)was hydrogenated with H₂ for 4 h at room temperature. The reactionmixture was then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 5as a colorless oil (4.2 g, 38%). [M+Na]⁺=440.7

tert-butyl(R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-5-fluorophenoxy)acetate(6). A solution of ketone 5 (4.2 g, 10 mmol) in dry THF (40 mL) at −20°C. was treated with a solution of (+)-DIPChloride (20 mmol) in heptane(1.7 M, 11.8 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 5, then quenched with2,2′-(ethylenedioxy)diethylamine (2.9 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:5) togive compound 6 as a light yellow oil (2.94 g, 70%, ee 98% vs racemate).[M+Na]⁺=443.0

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-5-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (1.8 g, 4.28 mmol) and 7 (2 g, 6.42 mmol) in CH₂Cl₂(18 mL) was cooled to −20° C. before a solution of DCC (1.33 g, 6.42mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solutionof 4-(dimethylamino)pyridine (DMAP, 52 mg, 0.43 mmol) in CH₂Cl₂ (2 mL)under argon atmosphere. The resulting white suspension was allowed tostir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:1) to give compound 8 as a lightyellow oil (2.7 g, 90%). [M+Na]⁺=735.9

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-5-fluorophenoxy)aceticacid (Rae19). A solution of 8 (2.7 g, 4.11 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae19 (1.094 g, 44%) as apale yellow solid.

FKBD Example 342-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)aceticacid (Rae20)

(E)-3-(3,4-dimethoxyphenyl)-1-(4-fluoro-3-hydroxyphenyl)prop-2-en-1-one(3). To the solution of 3,4-dimethoxybenzaldehyde 1 (6.391 g, 38.5 mmol)and 1-(4-fluoro-5-hydroxyphenyl)ethan-1-one 2 (5.39 g, 35 mmol) in EtOH(70 mL) was added a solution of 40% aqueous KOH (19.6 g, 140 mmol) at 0°C. The resulting solution was reacted at room temperature for 4 h. Theyellow solid was filtrated to give compound 3 (9.368 g, 89%).[M+H]⁺=303.2

tert-butyl(E)-2-(5-(3-(3,4-dimethoxyphenyl)acryloyl)-2-fluorophenoxy)acetate (4).A solution of 3 (9.368 g, 31 mmol) and K₂CO₃ (5.1 g, 37 mmol) in DMF (90mL) was treated with tert-butyl bromoacetate (7.2 g, 37 mmol) andallowed to stir at room temperature for 4 h. After this time thereaction mixture was quenched by H₂O and extracted with EtOAc twice. Thecombined organic layers were concentrated in vacuo, which was used forthe next step without purification (13 g, quant.). [M+Na]⁺=438.9

tert-butyl2-(5-(3-(3,4-dimethoxyphenyl)propanoyl)-2-fluorophenoxy)acetate (5). Asolution of 4 (13 g, 31.2 mmol) and 10% Pd/C (1.3 g) in THF (200 mL) washydrogenated with H₂ for 4 h at room temperature. The reaction mixturewas then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 5as a colorless oil (7 g, 54%). [M+Na]⁺=441.2

tert-butyl(R)-2-(5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-2-fluorophenoxy)acetate(6). A solution of ketone 5 (7 g, 16.7 mmol) in dry THF (40 mL) at −20°C. was treated with a solution of (+)-DIPChloride (33.5 mmol) in heptane(1.7 M, 19.7 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 5, then quenched with2,2′-(ethylenedioxy)diethylamine (4.89 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:5) togive compound 6 as a light yellow oil (4.9 g, 71%, ee 96% vs racemate).[M+Na]⁺=443.3

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-4-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (1.8 g, 4.28 mmol) and 7 (2 g, 6.42 mmol) in CH₂Cl₂(18 mL) was cooled to −20° C. before a solution of DCC (1.33 g, 6.42mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solutionof 4-(dimethylamino)pyridine (DMAP, 52 mg, 0.43 mmol) in CH₂Cl₂ (2 mL)under argon atmosphere. The resulting white suspension was allowed tostir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:1) to give compound 8 as a lightyellow oil (2 g, 65%). [M+Na]⁺=736.4

2-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)aceticacid (Rae20). A solution of 8 (1.8 g, 2.52 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae20 (835 g, 50%) as a paleyellow solid.

FKBD Example 352-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)aceticacid (Rae21)

(E)-3-(3,4-dimethoxyphenyl)-1-(2-fluoro-3-hydroxyphenyl)prop-2-en-1-one(3). To the solution of 3,4-dimethoxybenzaldehyde 1 (9.7 g, 58.4 mmol)and 1-(2-fluoro-3-hydroxyphenyl)ethan-1-one 2 (5.39 g, 35 mmol) in EtOH(70 mL) was added a solution of 40% aqueous KOH (19.6 g, 140 mmol) at 0°C. The resulting solution was reacted at room temperature for 4 h. Theyellow solid was filtrated to give compound 3 (3.5 g, 30%). [M+H]⁺=303.0

tert-butyl(E)-2-(3-(3-(3,4-dimethoxyphenyl)acryloyl)-2-fluorophenoxy)acetate (4).A solution of 3 (3.5 g, 11.6 mmol) and K₂CO₃ (1.92 g, 13.9 mmol) in DMF(40 mL) was treated with tert-butyl bromoacetate (2.7 g, 13.9 mmol) andallowed to stir at room temperature for 4 h. After this time thereaction mixture was quenched by H₂O and extracted with EtOAc twice. Thecombined organic layers were concentrated in vacuo, which was used forthe next step without purification (3.9 g, 80%). [M+H]⁺=416.9

tert-butyl2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)-2-fluorophenoxy)acetate (5). Asolution of 4 (3.5 g, 8.4 mmol) and 10% Pd/C (350 mg) in THF (50 mL) washydrogenated with H₂ for 4 h at room temperature. The reaction mixturewas then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 5as a colorless oil (2.45 g, 70%). [M+Na]⁺=441.0

tert-butyl(R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-2-fluorophenoxy)acetate(6). A solution of ketone 5 (2.45 g, 5.85 mmol) in dry THF (30 mL) at−20° C. was treated with a solution of (+)-DIPChloride (17.6 mmol) inheptane (1.7 M, 10.3 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 5, then quenched with2,2′-(ethylenedioxy)diethylamine (3 mL) by forming an insoluble complex.After stirring at RT for another 30 min, the suspension was filteredthrough a pad of celite and concentrated. The crude compound waspurified by silica-gel flash column chromatography (AcOEt/PE 1:5) togive compound 6 as a light yellow oil (2.3 g, 94%, ee >99%).[M+Na]⁺=443.0

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-2-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (1.728 g, 4.1 mmol) and 7 (1.919 g, 6.15 mmol) inCH₂Cl₂ (18 mL) was cooled to −20° C. before a solution of DCC (1.26 g,6.15 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 49 mg, 0.4 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:5) to give compound 8 as a lightyellow oil (2 g, 70%). [M+Na]⁺=735.7

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)aceticacid (Rae21). A solution of 8 (2 g, 2.52 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae21 (1.238 g, 67%) as awhite solid.

FKBD Example 362-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-hydroxyphenoxy)aceticacid (Rae24)

1-(4-(benzyloxy)-3-hydroxyphenyl)ethan-1-one (2). A solution of 1(19 g,125 mmol) and K₂CO₃ (17.2 g, 125 mmol) in DMF (250 mL) was treated withbenzyl bromide (21.2 g, 125 mmol) and allowed to stir at roomtemperature for 12 h. After this time the reaction mixture was pouredinto ice, yellow solid was precipitated. The mixture was filtrated andthe solid was washed with water (300 mL) to give 2 (13 g, 43%) as awhite solid. [M+H]⁺=243.1

(E)-1-(4-(benzyloxy)-3-hydroxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one(4). To the solution of 2 (12.7 g, 52.47 mmol) and 3 (10.5 g, 62.97mmol) in EtOH (60 mL) was added a solution of 40% aqueous KOH (8.4 g,209.8 mmol) at 25° C. The resulting solution was heated to 45° C. for 8h. The solution was adjusted to pH 4 by added 4M aqueous HCl dropwise at0° C., generated a large of yellow solid. Then the mixture was fiteredand the filter cake was washed with water (100 mL) to afford 4 (16.5 g,80%) as a yellow solid. [M+H]+=391.2.

tert-butyl(E)-2-(2-(benzyloxy)-5-(3-(3,4-dimethoxyphenyl)acryloyl)phenoxy)acetate(5). A solution of 4 (16.4 g, 42 mmol) and K₂CO₃ (11.6 g, 84.1 mmol) inDMF (50 mL) was treated with tert-butyl bromoacetate (12.23 g, 63.07mmol) and allowed to stir at room temperature for 12 h. After this timethe reaction mixture was poured into ice, yellow solid was precipitated.The mixture was filtered and the solid was washed with water (100 mL).The crude product was washed by petroleum ether (100 mL) to give 5 (18.5g, 88%) as a yellow solid. [M+H]⁺=504.9.

tert-butyl2-(5-(3-(3,4-dimethoxyphenyl)propanoyl)-2-hydroxyphenoxy)acetate (6). Asolution of 5 (18.0 g, 35.7 mmol) and 10% Pd/C (2 g) in THF (400 mL) washydrogenated with H₂ for 4 h at room temperature. The reaction mixturewas then filtered and concentrated. The crude product 6 (16 g, 88%) wasused to the next step directly. [M+Na]⁺=439.0

tert-butyl2-(2-((tert-butoxycarbonyl)oxy)-5-(3-(3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate(7). A solution of 6 (3 g, 7.2 mmol) and Boc₂O (2.35 g, 10.8 mmol) indry DCM (60 mL) at 25° C. was treated with DMAP (0.87 g, 7.2 mmol) at25° C. After stirring at room temperature for 1 h, the solution wasconcentrated in vacuum. The crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:3) to give compound 7 (2.5 g,67%) as a light yellow oil. [M+Na]⁺=538.9.

tert-butyl(R)-2-(2-((tert-butoxycarbonyl)oxy)-5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate(8). A solution of 7 (2.3 g, 4.45 mmol) in dry THF (20 mL) at −20° C.was treated with a solution of (+)-DIPChloride (13.3 mmol) in heptane(1.7 M, 8 mL) at −20° C. The resulting mixture was reacted at −20° C.until complete conversion of 7, then quenched with2,2′-(ethylenedioxy)diethylamine (1.97 g) by forming an insolublecomplex. After stirring at room temperature for another 30 min, thesuspension was filtered through a pad of celite and concentrated. Thecrude compound was purified by silica-gel flash column chromatography(AcOEt/PE 1:4) to give compound 8 (2 g, 86%) as a light yellow oil.[M+Na]⁺=540.9.

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-4-((tert-butoxycarbonyl)oxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(10). A solution of 8 (2 g, 3.86 mmol) and 9 (1.8 g, 5.79 mmol) inCH₂Cl₂ (15 mL) was cooled to −20° C. before a solution of DCC (1.19 g,5.79 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (47 mg, 0.38 mmol) in CH₂Cl₂ (2mL) under argon atmosphere. The resulting white suspension was allowedto stir at −20° C. for 2 h. The reaction mixture was then filtered,evaporated, and the crude compound was purified by silica-gel flashcolumn chromatography (AcOEt/PE 1:3) to give compound 10 (2.2 g, 70%) asa light yellow oil. [M+Na]⁺=833.8.

2-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-hydroxyphenoxy)aceticacid (Rae24). Condition 1: A solution of 10 (50 mg, 0.06 mmol) in CH₂Cl₂(2 mL) was treated with a solution of 20% TFA in CH₂Cl₂ (1 mL) at 0° C.The mixture stirred at room temperature for 1 h. LCMS analysis showed nodesired product and start material can be detected. Condition 2: Asolution of 10 (50 mg, 0.06 mmol) in HCOOH (1 mL) was stirred at roomtemperature for 1 h. LCMS analysis showed no desired product and startmaterial can be detected.

FKBD Example 372-((5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-yl)oxy)aceticacid (Rae26)

(E)-3-(3,4-dimethoxyphenyl)-1-(5-hydroxypyridin-3-yl)prop-2-en-1-one(3). To the solution of 3,4-dimethoxybenzaldehyde 1 (5.0 g, 30.1 mmol)and 1-(5-hydroxypyridin-3-yl)ethan-1-one 2 (4.95 g, 36.12 mmol) in EtOH(200 mL) was added a solution of 40% aqueous KOH (16.83 g,120 mmol) at0° C. The resulting solution was reacted at room temperature for 8 h,followed by dilution with EtOAc. The organic layer was washed by water,brine, dried over Na₂SO₄ and concentrated in vacuo. The residue waspurified by silica-gel flash column chromatography (AcOEt/PE 1:3) togive compound 3 as a colorless oil (6.8 g, 80%). [M+H]⁺=285.9

tert-butyl(E)-2-((5-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-3-yl)oxy)acetate (4).A solution of 3 (6 g, 21.03 mmol) and K₂CO₃ (3.5 g, 25.24 mmol) in DMF(150 mL) was treated with tert-butyl bromoacetate (4.93 g, 25.24 mmol)and allowed to stir at room temperature for 4 h. After this time thereaction mixture was quenched by H₂O and extracted with EtOAc twice. Theorganic layers were dried over Na₂SO₄ and concentrated in vacuo. Theresidue was purified by silica-gel flash column chromatography (AcOEt/PE1:5) to give compound 4 as a yellow oil (4.5 g, 54%). [M+H]⁺=399.9

tert-butyl2-((5-(3-(3,4-dimethoxyphenyl)propanoyl)pyridin-3-yl)oxy)acetate (5). Asolution of 4 (4.5 g, 11.26 mmol) and 10% Pd/C (400 mg) in THF (100 mL)was hydrogenated with H₂ for 6 h at room temperature. The reactionmixture was then filtered and concentrated. The residue was purified bysilica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 5as a yellow oil (2.5 g, 56%) [M+H]⁺=402.2

tert-butyl(R)-2-((5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-3-yl)oxy)acetate(6). A solution of ketone 5 (2.5 g, 6.23 mmol) in dry THF (40 mL) at−20° C. was treated with a solution of (+)-DIPChloride (24.9 mmol) inheptane (1.7 M, 14.7 mL) at −20° C. The resulting mixture was reacted at−20° C. until complete conversion of 5, then quenched with2,2′-(ethylenedioxy)diethylamine (3.7 mL) by forming an insolublecomplex. After stirring at RT for another 30 min, the suspension wasfiltered through a pad of celite and concentrated. The crude compoundwas purified by silica-gel flash column chromatography (AcOEt/PE 1:5) togive compound 6 as a colorless oil (2 g, 80%, ee>99%). [M+H]⁺=404.0

(R)-1-(5-(2-(tert-butoxy)-2-oxoethoxy)pyridin-3-yl)-3-(3,4-dimethoxyphenyl)propyl(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate(8). A solution of 6 (1.898 g, 4.7 mmol) and 7 (2.196 g, 7.1 mmol) inCH₂Cl₂ (18 mL) was cooled to −20° C. before a solution of DCC (1.46 g,7.1 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of asolution of 4-(dimethylamino)pyridine (DMAP, 61 mg, 0.5 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension wasallowed to stir at −20° C. for 2 h. The reaction mixture was thenfiltered, evaporated, and the crude compound was purified by silica-gelflash column chromatography (AcOEt/PE 1:7) to give compound 8 as a lightyellow oil (2.05 g, 63%). [M+H]⁺=696.8

2-((5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-yl)oxy)aceticacid (Rae26). A solution of 8 (2 g, 2.87 mmol) in CH₂Cl₂ (12 mL) wastreated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. Themixture was allowed to react at room temperature until completeconversion. The reaction mixture was charged to silica-gel flash columndirectly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae26 (545.8 g, 30%) as awhite solid.

Linker Example 1

(Z)-hex-3-ene-1,6-diol (1). Hex-3-yne-1,6-diol (2.0 g), quinoline (0.12g) and Lindlar catalyst (0.30 g) were suspended in MeOH (15 mL).Hydrogen was filled in to the flask with a Schlenk line and a positivepressure was maintained with a balloon of hydrogen. The reaction wasstirred at RT for 12 h before filtered and concentrated. The crudeproduct (2.1 g) was co-evaporated with toluene (20 mL×2) to remove theresidue of MeOH. The product 1 was used without further purification.

(Z)-6-hydroxyhex-3-en-1-yl 4-methylbenzenesulfonate (2). Monotosylationof diol was obtained by a reported Ag₂O-assisted method (10). Thepercentage yield of monotosylation is 90% for cis-C₆ linker on 2.0 gscale. ¹H NMR (500 MHz, CDCl₃) δ 7.79 (d, J=8.3 Hz, 2H, aromatic), 7.35(d, J=8.0 Hz, 2H, aromatic), 5.63-5.48 (m, 1H, ═CH), 5.48-5.33 (m, 1H,═CH), 4.04 (t, J=6.7 Hz, 2H, OCH2), 3.64 (dd, J=12.3, 6.2 Hz, 2H, OCH2),2.45 (s, 3H, CH3), 2.44 (q, J=6.5 Hz, 2H), 2.28 (q, J=6.5 Hz, 2H). ¹³CNMR (126 MHz, CDCl₃) δ 129.86 (aromatic), 129.51 (aromatic), 127.93(═CH), 126.19 (═CH), 69.99 (OCH₂), 61.99 (OCH2), 30.89, 27.26, 21.69(CH₃). HRMS for [M+H]⁺ C13H₁₈O4S, calculated: 271.1004, observed:271.1004.

(3). To conjugate the Ts-protected alcohol on 2-chlorotrityl chloridesolid support, briefly, the resin (9.6 mmol, 1.14 mmol/g),2,6-di-tert-butylpyridine (10.5 mmol) and alcohol (5.9 mmol) was mixedin 100 mL CH₂Cl₂. AgOTf (10.0 mmol) was added in two aliquots over 15min. The red color of the resin persisted and this indicates that thealcohol is depleted in the reaction mixture. MeOH (5 mL) was then addedto quench the reaction and the color turned white or pale yellow over 5min. The suspension was stirred at RT for another 1 h before it wasfiltered and the solid-support was transferred to a separatory funnelwith CCl₄. After the mixture standing for 5 min to allow stratification,AgCl precipitation on the bottom was removed by draining the liquid to alevel that most floating resin remained. The resin was then collected ina 250 mL solid-support reactor and washed with pyridine (50 mL×4) withextensive shaking.

(cis-C6 linker). The resin was then transferred into a 250 mL RB-flaskwith 100 mL THF. Methylamine (33% in MeOH) was added and stirred at 40°C. for 12 h. The resin was filtered and washed with THF (50 mL) fortwice and CH₂Cl₂ (50 mL) for twice. For long time storage at −20° C.,the resin was further washed with MeOH and air-dried for 20 min. Themolarity of the NH group was determined by UV of the cleaved firstcoupled Fmoc group (0.40-0.45 mmol/g).

RAPAFUCIN EXAMPLES

General Automated Synthesis. Solid-phase peptide synthesis (SPPS) wereapplied with a split-pool strategy to assemble the tetrapeptide effectordomains. The pre-assembled FKBD capped with a carboxylic acid at one endand an olefin at the other was subsequently coupled to the tetrapeptidethat remained tethered on beads. To facilitate purification of the newlyformed macrocycles, we adopted a coupled macrocyclization and cyclativerelease strategy whereby the macrocyclization is accompanied by theconcurrent release of the macrocyclic products from the solid beads.After exploring different macrocyclization methods, ring-closingmetathesis/cyclative release (RCM) can be used for efficient parallelsynthesis of different Rapafucins. Both aFKBD and eFKBD possess highaffinity for FKBP12, with K_(d) values of 4 and 11 nM, respectively.Importantly, this enhanced affinity was largely retained onincorporation into macrocycles, with average Ka values of 25 and 37 nM,respectively. Moreover, there was relatively low variation in bindingaffinity for FKBP12 among different macrocycles bearing aFKBD or eFKBD.These results suggested that both aFKBD and eFKBD are tolerant todifferent effector domain sequences, thus rendering them suitable FKBDbuilding blocks for Rapafucin libraries.

Charged resin (4.800 g) was dissolved in DMF/DCM (1/4, v/v) anddispersed to each well of an Aapptec Vantage automated synthesizer (96wells). Wells were drained and swelled with DMF for 20 mins before thesolvent was drained and washed with 1× DMF. Fmoc-protected amino acidbuilding blocks (3.0 eq., —0.3M in DMF), HATU (3.0 eq., ˜0.1M in DMF),and DIEA (6 eq., ˜0.3M in DMF) were added in order to each of the 96wells. The resin and reagent mixture were mixed on the automatedsynthesizer for 2-3 hrs, then washed with DMF (5×) for 5 times. Ifcoupling was difficult, the coupling reaction would be repeated. Resinswere washed thoroughly with DMF (3×) for 3 times. Deprotection of theFmoc group was achieved by shaking resins with 1 mL of piperidine/DMF(1/4, v/v) for 10 min and 1 mL piperidine/DMF (1/4, v/v) for 5 min.Resins were washed thoroughly with DMF 5 times. Coupling reaction wasrepeated 4 times to achieve the synthesis of tetrapeptide. Couplingreactions were repeated if Fmoc-valine or -isoleucine were to be coupledto N-methyl amino acids on resin or if Fmoc-proline was used. Then thedeprotection of Fmoc group is performed. FKBD (3 eq., ˜0.2 M in DMF),HATU (3 eq., ˜0.1M in DMF), and DIEA (6 eq., ˜0.3M in DMF) were added inorder into the vessel of the prepared resin. The resin and reagentmixture were mixed on the automated synthesizer for 3 hrs, then washedwith DMF (2×) for 2 times and DCM (2×) for 2 times. 1.25 mL of EthylAcetate and 0.25 mL of Hoveyda-Grubbs II (30 mol %) were added to eachwell. The reaction block was 80° C. for 5 hrs. Upon reaction completion,the resulting brown suspension was purified on 1 g solid phaseextraction columns packed with 1 g silica gel. The columns were washedusing dichloromethane and eluted with 10% methanol in dichloromethane.The eluate was concentrated under vacuum and weighted. The compoundswere characterized using LC/MS analysis.

TABLE 9 Synthesis and characterization of compounds 1066, 1081, 1082,1087, 1088, and 1522. Composition (FKBD/ monomer1/ Com- monomer2/ Mole-Reten- pound monomer3/ cular tion Uptake, No. monomer4) weight time 293TMolecular Structure 1087 aFKBD ra602 ra140 dp ml 1289.54 3.92 low

1088 aFKBD ra348 mf dp ml 1276.54 4.11 low

1081 aFKBD ra602 ra553 dp ml 1338.61 4.24 medium

1082 aFKBD ra602 ra73 dp ml 1330.59 4.22 low

1522 aFKBD ra602 y dp ml 1240.46 3.65 high

1066 aFKBD ra602 ra559 dp ml 1262.51 4.07 high

General Manual Synthesis. Synthesized as previously described. (Guo etal. (2018) Nat. Chem. 11:254-63).

TABLE 10 Synthesis and characterization of compounds 560-574, 576, and1563-65. Composition (FKBD/ monomer1/ Com- monomer2/ Mole- Reten- poundmonomer3/ cular tion Prolif, No. monomer4) weight time A549 ChemicalStructure 560 rae1 ra147 napA ra562 g 1247.49 5.56 medium

561 rae2 ra147 napA ra562 g 1247.49 5.63 medium

562 rae3 ra147 napA ra562 g 1247.49 5.48 medium

563 rae4 ra147 napA ra562 g 1247.49 5.47 low

564 rae5 ra147 napA ra562 g 1247.49 5.48 low

565 rae9 ra147 napA ra562 g 1233.47 5.35 medium

566 rae10 ra147 napA ra562 g 1233.47 5.10 medium

567 rae11 ra147 napA ra562 g 1233.47 5.11 medium

568 rae12 ra147 napA ra562 g 1235.46 5.74 medium

569 rae13 ra147 napA ra562 g 1235.46 5.27 medium

570 rae14 ra147 napA ra562 g 1235.46 5.72 medium

571 rae16 ra147 napA ra562 g 1440.70 5.93 low

572 rae17 ra147 napA ra562 g 1232.48 4.41 medium

573 rae18 ra147 napA ra562 g 1235.46 5.49 low

574 rae19 ra147 napA ra562 g 1235.46 5.60 low

576 rae20 ra147 napA ra562 g 1235.46 5.56 medium

1563 rae21 ra147 napA ra562 g 1235.46 6.94 high

1564 rae29 ra147 napA ra562 g 1204.44 6.67 high

1565 rae26 ra147 napA ra562 g 1218.46 low

TABLE 11 Synthesis and characterization of compounds 1566-84.Composition (FKBD/ monomer1/ monomer2/ Compound monomer3/ MolecularProlif, No. monomer4) weight H929 Chemical Structure 1566 rae1 my df sardf 1251.44 medium

1567 rae10 my df sar df 1237.41 medium

1568 rae11 my df sar df 1237.41 low

1569 rae12 my df sar df 1239.41 low

1570 rae13 my df sar df 1239.41 medium

1571 rae14 my df sar df 1239.41 low

1572 rae16 my df sar df 1444.65 low

1573 rae16a my df sar df 1222.40 low

1574 rae17 my df sar df 1236.43 low

1575 rae18 my df sar df 1239.41 low

1576 rae19 my df sar df 1239.41 medium

1577 rae2 my df sar df 1251.44 medium

1578 rae20 my df sar df 1239.41 low

1579 rae21 my df sar df 1239.41 medium

1580 rae26 my df sar df 1222.40 low

1581 rae3 my df sar df 1251.44 medium

1582 rae4 my df sar df 1251.44 low

1583 rae5 my df sar df 1251.44 low

1584 rae9 my df sar df 1237.41 low

TABLE 12 Synthesis and characterization of compounds 1555-1557.Composition (FKBD/ monomer1/ Com- monomer2/ Mole- Reten- pound monomer3/cular tion Uptake, No. monomer4) weight time 293T Chemical Structure1555 raa18 ra602 mf dp ml 1237.51 4.39 high

1556 rae27 ra602 mf dp ml 1211.46 5.02 low

1557 raa17 ra602 mf dp ml 1237.51 4.37 high

Post cyclization modification. Protecting groups may be removed beforefinal purification. In some embodiments, a tert-butyl protecting groupcan be removed using TFA. A solution of protected Rapafucin is dissolvedin DCM and triethylsilane (2 Eq) is added. TFA (20% final concentration)is added and stirred for 2 hours. The mixture is reduced under vacuumand purified via normal phase chromatography (1:9 MeOH/DCM) to give ayellow solid. The compound is further reunified using reverse phasechromatography (40→95% ACN/H₂O ) to give a pale colored solid.

In some embodiments, a tert-butyloxycarbonyl protecting group may beremoved using TFA. A solution of protected Rapafucin is dissolved in DCMand triethylsilane (2 Eq) is added. TFA (20% final concentration) isadded and stirred for 2 hours. The mixture is reduced under vacuum andpurified via normal phase chromatography (1:9 MeOH/DCM) to give a yellowsolid. The compound is further reunified using reverse phasechromatography (40→95% ACN/H₂O) to give a pale colored solid.

Additional functional groups can be added to deprotected Rapafucins. Insome embodiments, reactive functional groups can be deprotected toproduce a chemical handle for additional modifications. These reactionsinclude substitution, addition, and radical reactions.

In some embodiments, a carbamate group is appended to an alcoholcontaining rapafucin. Other functional groups would work as well. Thisis an example of attaching an electrophile to the exposed nucleophile,in this embodiment, a phenol group. A deprotected alcohol (or phenol)containing Rapafucin is dissolved in DCM, then pyridine (10 mol %) andDIEA (3 Eq) was added. A solution of carbonyl chloride (3 Eq) in DCM wasadded dropwise and stirred for 2 hours. The solution was washed with asaturated ammonium chloride solution (3×) and dried over Mg₂SO₄. Thesolution concentrated and purified via column chromatography (0→20%MeOH/EtOAc) to produce a white solid.

TABLE 13 Synthesis and characterization of compounds 867-869 and 877.Composition (FKBD/ monomer1/ Com- monomer2/ Mole- Reten- pound monomer3/cular tion Prolif. No. monomer4) weight time H929 Chemical Structure 877rae37 ra398 df sar df 1319.52 4.181 low

867 rae21 ra492 df sar df 1352.52 5.75 low

868 rae19 ra492 df sar df 1352.52 5.54 low

869 aFKBD ra492 df sar df 1375.58 5.403 low

In some embodiments, an amide group is formed from an amine containingRapafucin. A deprotected amine containing Rapafucin is dissolved in DCM,then acyl chloride (2 Eq) and DIEA (3 Eq) was added. The solution waswashed with brine (3×) and dried over Mg₂SO₄. The solution concentratedand purified via column chromatography (0→20% MeOH/EtOAc) to produce awhite solid.

TABLE 14 Synthesis and characterization of compounds 1585-1589.Composition (FKBD/ monomer1/ Com- monomer2/ Mole- Reten- pound monomer3/cular tion Uptake, No. monomer4) weight time 293T Chemical Structure1585 afkbd phg ra655 dp ml 1357.60 3.72 High

1586 afkbd phg ra656 dp ml 1370.70 3.74 Med

1587 afkbd phg ra626 dp ml 1338.60 3.15 Low

1588 afkbd phg ra592 dp ml 1281.52 3.44 High

1589 afkbd phg ra618 dp ml 1358.60 3.10 Low

In some embodiments, an amide group is formed from carboxylic acidcontaining rapafucin. A deprotected carboxylic acid containing Rapafucinis dissolved in ethyl acetate (5 mM), then an amine (2 Eq), DIEA (10Eq), and T3P (2 Eq) was added. The reaction until the reaction wascomplete via LC/MS. The solution was washed with brine (3×) and theorganic layer was dried over Mg₂SO₄. The solution concentrated andpurified via column chromatography (0→20% MeOH/EtOAc) to produce a whitesolid.

TABLE 15 Synthesis and characterization of compounds 1558, 1559, 1562,1590, and 1591. Composition (FKBD/ monomer1/ Com- monomer2/ Mole- Reten-pound monomer3/ cular tion Uptake, No. monomer4) weight time 293TChemical structure 1558 afkbd phg ra500 dp ml 1311.50 3.81 high

1559 afkbd phg ra501 dp ml 1343.60 3.86 medium

1562 afkbd phg ra504 dp ml 1344.60 3.22 low

1590 afkbd phg ra620 dp ml 1371.64 3.919 Low

1591 afkbd phg ra623 dp ml 1365.68 3.956 Low

In some embodiments, a phosphinate group may be added to a rapafucin. Adeprotected alcohol (or phenol) containing Rapafucin is dissolved in DCMand pyridine (1:1 v/v) and dimethylphosphinic chloride (11 Eq) at roomtemperature and stirred for 16 hrs. The reaction mixture was dilutedwith DCM and washed with dilute HCl. The organic fraction was washedwith water and dried over Mg2SO₄. The solution concentrated and purifiedvia column chromatography (0420% MeOH/EtOAc) to produce a white solid.

TABLE 16 Synthesis and characterization of compound 1520. Composition(FKBD/ monomer1/ Com- monomer2/ Mole- Reten- pound monomer3/ cular tionUptake, No. monomer4) weight time 293T Chemical structure 1520 aFKBDra602 ra515 dp ml 1316.4 5.34 low

Manual Gram Scale Ring-Closing Metathesis. Charged Resin (LoadingCapacity=0.2-0.3 mmol/g) is loaded in a 500 ml of SPPS vessel andswelled for 30 min with DCM (300 ml) on laboratory shaker (Kamush®LP360AMP, 360°, speed 6), then filtered and washed with DMF (200 ml×2)and dried under vacuum for 5 min.

A solution of Fmoc-AA (3 eq) and HATU (3 eq) in 150 ml of DMF was addedto the resin. Then DIEA (6 eq) in 50 ml of DMF was added and shaken for3 hrs. Solvent was filtered and washed with DMF (200 ml ×5) and DCM (200ml×5) and dried. 300 ml of 20% Piperidine in DMF was added and shakenfor 20-30 min, filtered and again 300 ml of 20% Piperidine in DMF wasadded and shaken for 20-30 min. The solvent was filtered and washedcarefully with DMF (200 ml×5), then immediately taken for next Fmoc-AAcoupling.

After the peptidic portion is installed and deprotected, FKBD (2 eq) wasalso coupled similar manner was taken for next step (No de-protection ofthe FKBD necessary). LC-MS analysis was performed after every Fmoc-AAcoupling.

Linear Rapafucin on resin and Hoveyda-Grubbs II (30 mol %) was taken ina 2 L round bottom flask with 8cm long octagonal stir bar. Ethyl acetate(600 mL) was taken in 2 L conical flask and sparged with gentle streamof N₂ for —10 min, then was added to the Resin/Catalyst mixture. A superair condenser was mounted and the flask was placed in oil bath andheated to 90° C. for 5 h (moderate reflux) under N₂ (Balloon). Thesolution was cooled to room temperature leaving a dark brown solutionwith suspended resin. The resin was checked using LC/MS and TLC forformation of desired product.

Resin was filtered off and the filtrate was evaporated in vacuo togenerate a dark brown crude product which was dissolved in minimal DCM(60 mL) and subjected into normal phase column chromatography (0→10%MeOH/EtOAc). Fractions containing pure desired compound were pooled andconcentrated in vacuo to yield a brownish powder. The product was thendissolved in a minimal amount of MeOH (20 mL) and subjected into reversephase column chromatography (10 to 95% ACN/H₂O). Fractions containingpure desired compound were pooled and concentrated in vacuo to getoff-white solid, which was dissolved in 20-25 ml of 2-MeTHF and drippedinto the 250 ml of Heptane in a 1 L flask with gentle stirring. Formedwhite precipitate was filtered and dried to get pale grayish whitepowder.

TABLE 17 Synthesis and characterization of compound 1592. Composition(FKBD/ monomer1/ Com- monomer2/ Mole- Reten- pound monomer3/ cular tionA549 No. monomer4) weight time Prolif Molecular Structure 1592 aFKBD mldf mi g 1178.44 6.48 High

Ring Closing via Macrolactamization. Unmodified 2-chloro-chlorotritylresin (Loading Capacity=1.5 mmol/g) is loaded into a solid phasereaction vessel (60 mL) and peptidic portion is synthesized under normalsolid phase synthesis conditions. (see above section).

For peptide residues that need alternative coupling conditions forracemization, the resin may be treated to the following conditions:Deprotected resin is cooled to 0° C. Resin was treated with a cold (0°C.) pre-mixed (5 minutes) solution of FMOC-Amino Acid (3 Eq) in DMF,Oxyma (3 Eq) in DMF and DIEA (3 Eq); shaken for 3 hours. The resultantresin was filtered and washed with DMF (5×3 ml), DCM (5×3 ml) and dried.

After deprotection of the peptidic portion on resin, a FKBD containing aprotected amine functionality can be installed using normal syntheticprocedures. The resultant fragment can be deprotected and released fromthe resin.

The FKBD containing linear rapafucin can be further cyclized to producethe cyclic Rapafucin. Acyclic Rapafucin is taken up in DMF and treatedwith COMU-PF6 (3 Eq) and DIEA (3 Eq), let stir for 1 hour. The reactionis monitored by LC/MS. Upon completion, the mixture is diluted withwater and extracted with EtOAc (3×). Combined extracts were washed withbrine, dried over MgSO₄ and reduced under vacuum. The crude product ispurified via column chromatography (1:9 MeOH/EtOAc) to give an orangesolid and repurified via reverse phase chromatography (40→95% ACN/H₂O)to give a tan solid.

If required protecting groups may be removed before final purification.In some embodiments, a tert-butyl protecting group can be removed usingTFA. A solution of protected Rapafucin is dissolved in DCM andtriethylsilane (2 Eq) is added. TFA (20% final concentration) is addedand stirred for 2 hours. The mixture is reduced under vacuum andpurified via normal phase chromatography (1:9 MeOH/DCM) to give a yellowsolid. The compound is further reunified using reverse phasechromatography (40→95% ACN/H₂O) to give a pale colored solid.

PROPHETIC EXAMPLES—DNA-ENCODED LIBRARY Prophetic Example 1—Preparationof a Rapafucin DNA-Encoding Library via Split-and-Pool Cycles

A rapafucin DNA-encoding library is synthesized by a sequence ofsplit-and-pool cycles wherein the oligonucleotide is attached to theFKBD. First, an initial oligonucleotide of Formula (XIII) is synthesizedand HPLC purified. A first building block comprising an FKBD buildingblock is then covalently bound to the oligonucleotide of Formula (XIII)via click chemistry. Subsequently, a second oligonucleotide, encodingthe first building block, is appended to the oligonucleotide of Formula(XIII). The resulting product is pooled and split into a second set ofseparate reaction vessels and a second building block comprising aneffector domain building block is coupled to the first building blockusing a ring-closing reaction. The reaction is then encoded by theattachment of a unique oligonucleotide sequence to the uniqueoligonucleotide attached to the first building block. The encodedtwo-building-block molecules yields the final library.

Prophetic Example 2—Preparation of a Rapafucin DNA-Encoding Library viaSplit-and-Pool Cycles

A rapafucin DNA-encoding library is synthesized by a sequence ofsplit-and-pool cycles wherein the oligonucleotide is attached to alinking region. First, an initial oligonucleotide of Formula (XIII) issynthesized and HPLC purified. Then, the oligonucleotide of Formula(XIII) is covalently bound to a first linking region via clickchemistry. A first building block comprising an FKBD building block isencoded by a second oligonucleotide which is appended to the initialoligonucleotide of Formula (XIII). The resulting product is pooled andsplit into a second set of separate reaction vessels and a secondbuilding block comprising an effector domain building block is coupledto the first building block using a ring-closing reaction. The reactionis then encoded by the attachment of a unique oligonucleotide sequenceto the unique oligonucleotide attached to the first building block. Theencoded two-building-block molecules yields the final library.

Prophetic Example 3—Preparation of a Rapafucin DNA-Encoding Library viaDNA-Recorded Synthesis and Ligation

A rapafucin DNA-encoding library is synthesized by DNA-recordedsynthesis wherein the oligonucleotide is attached to the FKBD. First, aninitial oligonucleotide of Formula (XIII) is synthesized and HPLCpurified. A first building block comprising an FKBD building block isthen covalently bound to the oligonucleotide of Formula (XIII) via clickchemistry. Then, a second building block comprising an effector domainbuilding block is coupled to the first building block via the first andsecond linking region through a ring-closing reaction. The reaction isencoded by DNA-recorded synthesis by ligation of a uniqueoligonucleotide to the initial oligonucleotide of formula (XIII).

Prophetic Example 4—Preparation of a Rapafucin DNA-Encoding Library viaDNA-Recorded Synthesis and Enzymatic Reactions

A rapafucin DNA-encoding library is synthesized by DNA-recordedsynthesis wherein the oligonucleotide is attached to the FKBD. First, aninitial oligonucleotide of Formula (XIII) is synthesized and HPLCpurified. A first building block comprising an FKBD building block isthen covalently bound to the oligonucleotide of Formula (XIII) via clickchemistry. Then, a second building block comprising an effector domainbuilding block is coupled to the first building block via the first andsecond linking region through a ring-closing reaction. The reaction isthen encoded by DNA-recorded synthesis by polymerase-catalyzed fill-inreactions.

Prophetic Example 5—Preparation of a Rapafucin DNA-Encoding Library viaDNA-Templated Synthesis

A rapafucin DNA-encoding library is synthesized by DNA-temapltedsynthesis. First, a second building block comprising an effector domainbuilding block is coupled to the first building block comprising theFKBD via the first and second linking regions. Then, the reaction isencoded by DNA-templated synthesis, wherein a plurality of conjugatemolecules of oligonucleotide-tagged building blocks are prepared and thespatial proximity of the two distinct oligonucleotides of Formula (XIII)facilitates the bimolecular chemical reactions between the two buildingblocks.

EXAMPLES—BIOLOGICAL ASSAYS

Nucleoside Uptake Assay (uptake). Nuceloside uptake assays wereperformed with using 3H-Thymidine as described in Guo et al. (2018) Nat.Chem. 11:254-63. Specific cell lines are indicated in each assay andcultured in complete growth media. Activity is scored according to theIC₅₀ values relative to DMSO control. “Low” indicates an IC₅₀ greaterthan 600 nM, “Medium” indicates an IC₅₀ between 300 nM and 600 nM “High”indicates an IC₅₀ less than 300nM. “Rel.Uptake” refers to uptakeactivity characterization relative to a single concentration assay.“Low” indicates a response greater than 0.6 times the activity relativeto DMSO, “Medium” indicates a response between 0.6 and 0.3 times theactivity relative to DMSO, “High” indicates a response less than 0.3times the activity relative to DMSO.

Cell Proliferation Assay (Prolif) Guo et al. (2018) Nat. Chem.11:254-63. Specific cell lines are indicated in each assay and culturedin complete growth media. Activity is scored according to the IC₅₀values relative to DMSO control. “Low” indicates an IC₅₀ greater than600 nM, “Medium” indicates an IC₅₀ between 300 nM and 600 nM “High”indicates an IC₅₀ less than 300nM. “Rel.Uptake” refers to uptakeactivity characterization relative to a single concentration assay.“Low” indicates a response greater than 0.6 times the activity relativeto DMSO, “Medium” indicates a response between 0.6 and 0.3 times theactivity relative to DMSO, “High” indicates a response less than 0.3times the activity relative to DMSO.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific composition and procedures described herein. Such equivalentsare considered to be within the scope of this disclosure, and arecovered by the following claims.

What is claimed is:
 1. A compound of Formula (V)

or an optically pure stereoisomer or pharmaceutically acceptable saltthereof, wherein L is selected from the groups in Table 1; A is CH₂, NH,NMe, O, S(O)₂ or S; each X is independently O, NMe, or NH; E is CH or N;each of R¹, R², R³, and R⁴ is independently selected from the groupconsisting of H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂,COC₁₋₂₀alkyl, CO₂C₁₋₂₀alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₁₋₁₀alkoxy, C₆₋₁₅aryl, C₆₋₁₅aryloxy, C₆₋₁₅arylthio, C₂₋₁₀carboxyl,C₁₋₁₀alkylamino, thiol, C₁₋₁₀alkylthio, C₆₋₁₅arylthio,C₁₋₁₀heteroarylthio, (C₃₋₈cycloalkyl)thio, C₂₋₁₀heterocyclylthio,sulfonyl, C₁₋₁₀alkylsulfonyl, amido, C₁₋₁₀alkylamido, selenol,C₆₋₁₅arylselenol, C₁₋₁₀heteroarylselenol, (C₃₋₈cycloalkyl)selenol,C₂₋₁₀heterocyclylselenol, guanidino, C₁₋₁₀alkylguanidino, urea,C₁₋₁₀alkylurea, ammonium, C₁₋₁₀alkylammonium, cyano, C₁₋₁₀alkylcyano,C₁₋₁₀alkylnitro, adamantine, phosphonate, C₁₋₁₀alkylphosphonate, andC₆₋₁₅arylphosphonate, each of the above can be optionally substitutedwith H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₂₀alkyl, substitutedC₁₋₂₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino,acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl,aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino,aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl,C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substitutedC₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino,(carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl,(C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy,(C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl,C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy,substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substitutedC₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl,C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl; or R⁴ forms a cyclic structure formedwith R³, the cyclic structure is selected from the group consisting ofC₂₋₁₀heterocyclyl and C₁₋₁₀heteroaryloptionally substituted with H,halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substitutedC₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino,acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl,aminocarbonyl aminocarbonylamino, aminodicarbonylamino,aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl,C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substitutedC₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino,(carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl,(C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy,(C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl,C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy,substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substitutedC₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl,C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy,C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo,sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substitutedC₁₋₁₀alkylthio, and thiocarbonyl n is an integer selected from 0 to 4; mis an integer selected from 0 to 5; p is an integer selected from 0 to2; and q is an integer selected from 1 to
 10. 2. The compound of claim1, wherein q is 3 or
 4. 3. The compound of claim 1, wherein m is
 2. 4.The compound of claim 1, wherein R² is methoxy.
 5. The compound of claim1, wherein A is CH₂.
 6. The compound of claim 1, wherein R¹ is H and/orn is
 0. 7. The compound of claim 1, wherein E is CH.
 8. A pharmaceuticalcomposition comprising the compound of claim 1 or an optically purestereoisomer or pharmaceutically acceptable salt and a pharmaceuticallyacceptable carrier.
 9. A method of making a macrocyclic molecule,comprising: synthesizing an FKBP-binding domain (FKBD)-containingprecursor with a first terminal moiety and a second terminal moiety;assembling an effector domain based on natural or unnatural amino acidsusing solid state peptide synthesis; bonding the effector domain withthe FKBD-containing precursor via the first terminal moiety; andobtaining the macrocylic molecule via a macrocyclization method bybonding the second terminal moiety and the effector domain.
 10. Themethod of claim 9, wherein the macrocyliczation method can be selectedfrom the group consisting of ring-closing metathesis,macrolactamization, and click chemistry.
 11. The method of claim 9,wherein the FKBD-containing precursor has a structure of Formula (XVI)

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinRing A is a 5-10 membered aryl substituted with 1-17 substituentsindependently selected from the group consisting of halo, alkyl, alkoxy,cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino,dialkylamino,

or heteroaryl or heterocycloalkyl, optionally substituted with 1-17substituents, each of the substituent is independently selected from thegroup consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano,haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; J is independently at each occurrence selected from thegroup consisting of —C(O)NR⁶—,

R⁶ is each hydrogen, alkyl, arylalkyl,

R^(N) is aryl, alkyl, or arylalkyl; R′ is hydrogen, alkyl, arylalkyl, orhaloalkyl; D is independently at each occurrence an oligonucleotide;L^(b) and L^(c) are independently at each occurrence selected from thegroup consisting of bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—,—(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—,optionally substituted (CH₂)_(n)C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionallysubstituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted(CH₂)nNR⁵C(O)C₁₋₆alkylene (CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted(CH₂)_(n)OC(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted(CH₂)_(n)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)n-, optionally substituted(CH₂)_(n)—S—C₁₋₆ alkylene (CH₂)_(n)—, and optionally substituted(CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or2 groups independently selected from the group consisting of halo,hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano,nitro, NHFmoc; each R⁵ is independently hydrogen, alkyl, arylalkyl,

or and

R^(N) is aryl, alkyl, or arylalkyl; X is O, S or NR⁸, R⁸ is hydrogen,hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ areeach independently hydrogen or alkyl; V¹ and V² are each independently

Ring B is a 4-10 membered heterocycloalkyl, optionally substituted with1-10 substituents, each of which is selected from the group consistingof hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy,alkylthio, oxo,

amino, alkylamino, dialkylamino, arylalkyl, wherein R¹² is aryl, alkyl,or arylalkyl; R¹³ is hydrogen, hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is each independentlyhydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl,arylalkyl, or heteroaryl; Z is bond,

R¹⁶ and R¹⁷ are each independently selected from the group consisting ofhydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio,amino, alkylamino, and dialkylamino; K is O, CHR¹⁸, CR¹⁸ , N, or andNR¹⁸, wherein R¹⁸ is hydrogen or alkyl; L^(a), L¹, L², L³, L⁴, L⁵, L⁶,L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹—, —SO—, —SO₂—,(CH₂)_(n—,)

or a linking group selected from Table 1; Ring C is a 5-6 memberedheteroaryl, optionally substituted with 1-4 substituents, each of whichis independently selected from the group consisting of hydrogen,hydroxyl, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio,amino, alkylamino, dialkylamino and

wherein each R¹⁹, R²⁰, and R²¹ is independently is selected from thegroup consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl; and n is 0, 1, 2, 3, 4, 5 or6.
 12. The method of claim 11, wherein Ring A is a 5-10 membered aryl.13. The method of claim 11, wherein Ring A is phenyl.
 14. The method ofclaim 11, wherein Ring A is phenyl substituted by two methoxy groups.15. The method of claim 11, wherein L1 is

and Ring C is phenyl.
 16. The method of claim 11, wherein

and Ring B is piperidine.
 17. The method of claim 9, wherein theeffector domain has a structure of Formula (XIIa)

wherein each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), andk^(i) is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e),X^(f), X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—,—S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-,—(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; each w isindependently 0, 1, or 2; each R¹, R^(1a), R^(1b), R^(1c), R^(1d),R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ is independentlyhydrogen, alkyl, arylalkyl or NR²⁵; R²⁵ is hydrogen, hydroxy, OR²⁶,NR²⁷R²⁸, alkyl, arylalkyl,

R^(N) is aryl, alkyl, or arylalkyl; R²⁶, R²⁷, and R²⁸ are eachindependently hydrogen or alkyl; and each R², R³, R^(2a), R^(3a),R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f),R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) isindependently selected from the group consisting of hydrogen, halo,amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted alkylamino,optionally substituted dialkylamino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted arylalkyl,optionally substituted heteroarylalkyl and


18. The method of claim 17, wherein k^(a)=k^(b)=k^(c)=1.
 19. The methodof claim 18, wherein k^(d)=k^(c)=k^(f)=k^(g)=k^(h)=k^(i)=0.
 20. A taggedcompound of macrocyclic compound according to Formula (XII):

or a pharmaceutically acceptable salt, solvate, or stereoisomer thereofwith a compound of Formula (XIV):Q′-L^(c)-D   Formula (XIV) wherein Ring A is a 5-10 membered aryl,cycloalkyl, heteroaryl or heterocycloalkyl, optionally substituted with1-17 substituents, each of which is independently selected from thegroup consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano,haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; L^(b) and L^(c) are independently selected from the groupconsisting of a bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—,—(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—,optionally substituted (CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionallysubstituted (CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionallysubstituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)C(O)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted(CH₂)_(n)—S—C₁₋₆ alkylene-(CH₂)_(n)—, and optionally substituted(CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or2 groups independently selected from the group consisting of of halo,hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano,nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl,arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; Q and Q′ are independentlyselected from the group consisting of —N₃, —C≡CH, NR⁶R⁷, —COOH, —ONH₂,—SH, —NH₂,

—(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl,arylalkyl, or haloalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen,hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ areeach independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionallysubstituted with 1-10 substituents, each of which is selected from thegroup consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano,haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen,hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is eachindependently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl,haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the groupconsisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl,cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸,CR¹⁸, N, and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl; L^(a), L¹, L², L³,L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹, —SO—,—SO₂—, —(CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6membered heteroaryl, optionally substituted with 1-4 substituents, eachof which is independently selected from the group consisting ofhydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano,alkylthio, amino, alkylamino, dialkylamino and

wherein R¹⁹ is selected from the group consisting of hydrogen, hydroxy,OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴are each independently hydrogen or alkyl; n is 0, 1, 2, 3, 4, 5 or 6;wherein the Effector Domain has Formula (XIIa):

each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i)is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f),X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—,—S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄)alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b),R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ isindependently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ ishydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a),R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f),R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) isindependently selected from the group consisting of hydrogen, halo,amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted alkylamino,optionally substituted dialkylamino, optionally substituted aryl,optionally substituted heteroaryl, optionally substituted arylalkyl,optionally substituted heteroarylalkyl, and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnaturalamino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10; or wherein theEffector Domain has Formula (XIIc):

each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7,8, 9, and 10; R²⁹ is hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³²are each independently hydrogen or alkyl; X³ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; or wherein the EffectorDomain has Formula (XIId):

X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; or wherein the EffectorDomain has Formula (XIIe):

R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted orunsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆)alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; or wherein the EffectorDomain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆)alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl;wherein each w is independently 0, 1, or 2; and provided that when RingA is

L^(a) is ethylene, X is O, W is

V¹ is

V² is

Z is

-L⁶-L⁷-L⁸- is

and -L¹-L²-L³-L⁴-L⁵- is not

D is an oligonucleotide; wherein Ring A is substituted with at least one

or at least one of R², R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c),R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h),R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring Csubstituted with at least one

or wherein at least one of the linking groups selected from Table 1 issubstituted with at least one